Novel Microbial Succinic Acid Producers and Purification of Succinic Acid

ABSTRACT

The present invention relates to bacterial strains, capable of utilizing glycerol as a carbon source for the fermentative production of succinic acid, wherein said strains are genetically modified so that they comprise a deregulation of their endogenous pyruvate-formate-lyase enzyme activity, as well as to methods of producing organic acids, in particular succinic acid, by making use of such microorganism. The present invention also relates to the downstream processing of the produced organic acids by cation exchange chromatography.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/201,547 which is a national stage application (under 35U.S.C. §371) of PCT/EP2010/051798, filed Feb. 12, 2010, which claimsbenefit of European application 09152959.4, filed Feb. 16, 2009;European application 09171250.5, filed Sep. 24, 2009; and U.S.Provisional Application 61/245,306, filed Sep. 24, 2009.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_Listing_PF61823_(—)02. The size of thetext file is 30 KB, and the text file was created on Jan. 7, 2014.

FIELD OF THE INVENTION

The present invention relates to a bacterial strain, capable ofutilizing glycerol as a carbon source for the fermentative production ofsuccinic acid, wherein said strains are genetically modified so thatthey comprise a deregulation of their endogenous pyruvate-formate-lyaseenzyme activity as well as to methods of producing organic acids, inparticular succinic acid by making use of such microorganism.

BACKGROUND OF THE INVENTION

The fermentative production of succinic acid (SA) from biomass hasalready drawn much attention because said acid represents an importantconstituent of synthetic resins or is a source of further valuablelow-molecular chemical compounds, in particular tetrahydrofuran (THF),1,4-butanediol (BDO), gamma-butyrolactone (GBL) and pyrrolidones(WO-A-2006/066839).

A SA-producing bacterium isolated from bovine rumen was described by Leeet al (2002a). The bacterium is a non-motile, non-spore-forming,mesophilic and capnophilic gram-negative rod or coccobacillus.Phylogenetic analysis based on the 16S rRNA sequence and physiologicalanalysis indicated that the strain belongs to genus Mannheimia as anovel species, and has been named Mannheimia succiniciproducens MBEL55E.Under 100% CO₂ conditions, it grows well in the pH range of 6.0-7.5 andproduces SA, acetic acid and formic acid at a constant ratio of 2:1:1.When M. succiniciproducens MBEL55E was cultured anaerobically underCO₂-saturation with glucose as carbon source, 19.8 g/L of glucose wereconsumed and 13.3 g/L of SA were produced in 7.5 h of incubation.Furthermore in this microorganism the production of SA was improved bymutation/deletion of metabolic genes. The combined mutation/deletion ofthe genes lactate dehydrogenase ldhA, pyruvate-formate-lyase pflb,phosphotransacetylase pta, and acetate kinase ackA genes resulted in astrain converting carbon to SA with a yield (YP/S) of 0.6 g SA per g ofcarbon source added. The space-time yield for the production of SA wasfound to be 1.8 g/liter/h. (Lee 2006)

Lin et al 2005 describe a mutant strain of E. coli carrying mutations inthe ldh as well as in the pfl genes, described as SB202. However thisstrain was characterized by slow growth and the inability to ferment asaccharide to completion under anaerobic conditions. Inactive ldh andpfl did cause the carbon flux to bottle up at the pyruvate node, causingpyruvate to accumulate as the major product. In this respect the carbonyield (YP/S) of succinate on the carbon source was found to be lowerthan 0.15 g/g SA/Carbon.

Sanchez et al. 2005 describe E. coli strains carrying mutations in theldh, the adhE, ack-pta and icIR genes. In these experiments cells weregrown aerobically on complex medium, harvested, concentrated andincubated with carbon sources under anaerobic conditions. Under thesespecific conditions for the direct conversion of a carbohydrate to SA,carbon yields YP/S of 0.98 to 1.13 g SA per g carbon source were found,with a space-time yield of 0.79 g/l h SA. The carbon utilization for thebiomass generation prior to the anaerobic conversion phase has beenexplicitly not included in this calculation and is not furtherdescribed.

Hong and Lee (2001) describe E. coli strains carrying mutations in theldh and pfl genes. These strains do produce SA from the fermentation ofcarbohydrate, however, with slow carbohydrate utilization and lowspace-time and carbon yields (YP/S) of SA from the carbohydrate carbonsource glucose. In addition succinic, acetic and lactic acid wereproduced in a ratio of 1:0,034:1.6. In this analysis the growth of thestrain carrying mutations in the ldh and pfl genes was retarded ifcompared to the unmutated parental strain.

Zhu et al. 2005 describe a E. coli strain, mutated in the pfl gene whichdid not produce succinic acid but lactate and showed poor growth whengrown on glucose as the sole substrate.

A significant drawback of the organism Mannheirnia succiniciproducensis, however, its inability to metabolize glycerol, which, as aconstituent of triacyl glycerols (TAGs), becomes readily available e.g.as by-product in the transesterification reaction of Biodieselproduction (Dharmadi et al., 2006).

The fermentative production of SA from glycerol has been described inthe scientific literature (Lee et al., 2001: Dharmadi et al., 2006) andwith glycerol higher yields [mass of SA produced/mass of raw materialconsumed] than with common sugars like glucose were achieved (Lee etal., 2001). However, the space-time yield obtained with glycerol wassubstantially lower than with glucose (0.14 vs. 1.0 g SA/[L h]).

Only in a few cases anaerobic metabolization of glycerol to fermentationproducts have been described. E. coli is able to ferment glycerol undervery specific conditions such as acidic pH, avoiding accumulation of thefermentation gas hydrogen, and appropriate medium composition (Dharmadiet al 2006, Yazdani and Gonzalez 2007). Many microorganisms are able tometabolize glycerol in the presence of external electron acceptors(respiratory metabolism), few are able to do so fermentatively (i.e. inthe absence of electron acceptors). The fermentative metabolism ofglycerol has been studied in great detail in several species of theEnterobacteriaceae family, such as Citrobacter freundii and Klebsiellapneumoniae. Dissimilation of glycerol in these organisms is strictlylinked to their capacity to synthesize the highly reduced product1,3-propanediol (1,3-PDO) (Dharmadi et al 2006). The conversion ofglycerol into SA using Anaerobiospirillum succiniciproducens has beenreported (Lee et al. 2001). This study demonstrated that SA could beproduced with little formation of by-product acetic acid by usingglycerol as a carbon source, thus facilitating purification of SA. Thehighest yield was obtained by intermittently feeding glycerol and yeastextract, a strategy that resulted in the production of about 19 g/L ofSA. It was noted, however, that unidentified nutritional componentspresent in yeast extract were needed for glycerol fermentation to takeplace. Saccharides, however, theoretically can be converted to SA with asignificantly lower yield than glycerol due to the lower reduction stateof saccharides compared to the polyol glycerol. The combination ofsaccharides with glycerol have been found to function in an SA producinganaerobic organisms (Lee et al. 2001), however without reaching SAtiters beyond 29 g/l. In addition the carbon yield YP/S of was found tobe only 92% and the SA/AA relation was found to be 4.9:1. Only 4 g/lglycerol were converted to succinic acid at most.

Carboxylation reactions of oxaloacetate catalyzed by the enzymesphopshoenolpyruvate carboxylase (PEPC), phopshoenolpyruvatecarboxykinase (PEPCK) and pyruvate carboxylase (PycA) are utilizingHCO₃— as a source of CO₂ (Peters-Wendisch, P G et al 1996, 1998).Therefore hydrogencarbonate sources such as NaHCO₃, KHCO₃, NH₄HCO₃ andso on can be applied to fermentation and cultivation media to improvethe availibility of HCO₃— in the metabolization of substrates to SA. Theproduction of SA from glucose has not been found to be dependent on theaddition of HCO₃— in the prior art so far.

Biomass production by anaerobic organisms is limited by the amount ofATP produced from fermentative pathways. Biomass yield of glycerol inanaerobic organisms is lower than of saccharides, like hexoses such asglucose, fructose, pentoses such as xylose arabinose or disaccharidessuch as sucrose or maltose (Lee et al. 2001, Dharmadi 2007).

Earlier patent application PCT/EP2008/006714, the content of which isherewith incorporated by reference, discloses a bacterial strain, beinga member of the family of Pasteurellaceae, originally isolated fromrumen, and capable of utilizing glycerol as a carbon source and variantand mutant strains derived there from retaining said capability, inparticular, a bacterial strain designated DD1 as deposited with DSMZ(Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Inhoffenstr. 7B, D-38124 Braunschweig, Germany) having the depositnumber DSM 18541 (ID 06-614) and having the ability to produce succinicacid and variant or mutant strains derived there from retaining at leastsaid ability to produce succinic acid. The DD1 strain belongs to thespecies Basfia succiniciproducens and the family of Pasteurellaceae asclassified by Kuhnert et al., 2010.

There is, therefore, a need for novel bacterial strains, which have theability to produce organic acids, in particular SA, from glycerol. Inparticular, such strains should produce said acids with highproductivity from glycerol, especially if crude glycerol e.g. from biodiesel production can be used without prior purification. It is anobject of the present invention to provide such novel strains andproduction processes.

SUMMARY OF THE INVENTION

The present inventors, who had isolated a bacterial strain, designatedDD1, surprisingly solved said object by mutating said strain, so thatthe activity of the PFL protein was decreased so that said strain hasthe desired metabolic characteristic. Thus, they provided a new type ofbacterial stain, capable of utilizing glycerol as a carbon source forthe fermentative production of succinic acid, wherein said strain isgenetically modified so that it comprises a deregulation of itsendogenous PFL enzyme activity.

The present inventors surprisingly found that such a mutated bacterialstrain, having the desired metabolic characteristic, showed largelyimproved technical behavior in the fermentation of SA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic map of plasmid pSacB (SEQ ID NO:3).

FIG. 2 depicts a schematic map of plasmid pSacB (delta pfl) (SEQ IDNO:4).

FIG. 3 depicts a schematic map of plasmid pSacB (delta ldh) (SEQ IDNO:5).

DETAILED DESCRIPTION OF THE INVENTION a) General Definition ofParticular Terms

The term “bacterial cell” as used herein refers to a prokaryoticorganism, i.e. a bacterium. Bacteria can be classified based on theirbiochemical and microbiological properties as well as their morphology.These classification criteria are well known in the art.

The term “acid” (in the context of organic mono or dicarboxylic acids asreferred to herein, i.p. acetic, lactic and SA) has to be understood inits broadest sense and also encompasses salts thereof, as for examplealkali metal salts, like Na and K salts, or earth alkali salts, like Mgand Ca salts, or ammonium salts; or anhydrides of said acids.

“Identity” or “homology” between two nucleotide sequences means identityof the residues over the complete length of the aligned sequences, suchas, for example, the identity calculated (for rather similar sequences)with the aid of the program needle from the bioinformatics softwarepackage EMBOSS(Version 5.0.0, webpage at emboss.sourceforge.net/what/)with the default parameters which are:

-   -   gapopen (penalty to open a gap): 10.0    -   gapextend (penalty to extend a gap): 0.5    -   datafile (scoring matrix file included in package): EDNAFUL

The term “bacterial strain containing a mutated gene coding for apyruvate-formate-lyase enzyme with decreased activity” encompasses amodified bacterial cell which has a decreased activity or even nodetectable PFL activity. Methods for the detection and determination ofPFL activity can be found in Knappe et al. 1990 and Knappe 1993 andreferences therein. Moreover, the term encompasses a bacterial cell,which has a significantly reduced PFL activity when compared to abacterial cell exhibiting physiological pyruvate-formate-lyase activitylevels. Whether a reduction is significant can be determined bystatistical methods well known to those skilled in the art. Bacterialcells being deficient in PFL activity may occur naturally, i.e. due tospontaneous mutations. A bacterial cell can be modified to lack or tohave significantly reduced PFL activity by various techniques.Preferably, such bacterial cells are obtainable by chemical treatment orradiation. To this end, bacterial cells will be treated by, e.g., amutagenic chemical agent, X-rays, or UV light. In a subsequent step,those bacterial cells which lack PFL or which at least have a reducedPFL activity will be selected. Bacterial cells are also obtainable byhomologous recombination techniques, which aim to mutate, disrupt orexcise the PFL in the genome of the bacterial cell or introducemutations which will lead to a mutated gene coding for a protein withdecreased activity. A preferred technique for recombination, inparticular for introducing mutations or for deleting sequences, isdescribed below.

The above definition also applies to other genes coding for anotherenzyme mentioned herein, to be modulated, in particular, whose activityis to be deceased, diminished or switched-off.

The term “decreased activity” includes for example the expression of agene product (e.g. pyruvate-formate-lyase (pfl), lactate dehydrogenase(ldh) or others) by said genetically manipulated (e.g., geneticallyengineered) microorganism at a lower level than that expressed prior tomanipulation of the microorganism. Genetic manipulation can include, butis not limited to, altering or modifying regulatory sequences or sitesassociated with expression of a particular gene (e.g., by removingstrong promoters, inducible promoters or multiple promoters), modifyingthe chromosomal location of a particular gene, altering nucleic acidsequences adjacent to a particular gene such as a sequence in thepromoter region including regulatory sequences important for thepromoter activity a ribosome binding site or transcription terminator,decreasing the copy number of a particular gene, modifying proteins(e.g., regulatory proteins, suppressors, enhancers, transcriptionalactivators and the like) involved in transcription of a particular geneand/or translation of a particular gene product, or any otherconventional means of decreasing expression of a particular gene routinein the art (including but not limited to use of antisense nucleic acidmolecules, or other methods to knock-out or block expression of thetarget protein).

In particular the gene can be manipulated that one or more nucleotidesare being deleted from the chromosome of the host organism. Thedecreased activity of the gene product e.g. of a pyruvate-formate-lyasemolecule, can also be obtained by introducing one or more gene mutationswhich lead to a decreased activity of the gene product. The decreasedactivity can be a reduction of the enzymatic activity by ≧50% of thenon-mutated or unaltered enzyme activity, or reduction of the enzymaticactivity by ≧90%, or more preferably a reduction of the enzymaticactivity by ≧95%, or more preferably a reduction of the enzymaticactivity by ≧98%, or even more preferably a reduction of the enzymaticactivity by ≧99% or even more preferably a reduction of the enzymaticactivity by ≧99.9%.

The term “recombinant” microorganism includes a microorganism (e.g.,bacteria, yeast cell, fungal cell, etc.) which has been geneticallyaltered, modified or engineered (e.g., genetically engineered) such thatit exhibits an altered, modified or different genotype and/or phenotype(e.g., when the genetic modification affects coding nucleic acidsequences of the microorganism) as compared to the naturally-occurringmicroorganism from which it was derived. The term “promoter” refers to aDNA sequence, which directs the transcription of a structural gene toproduce mRNA. Typically, a promoter is located in the 5′ region of agene, proximal to the start codon of a structural gene. If a promoter isan inducible promoter, then the rate of transcription increases inresponse to an inducing agent. In contrast, the rate of transcription isnot regulated by an inducing agent, if the promoter is a constitutivepromoter.

The term “enhancer” refers to a promoter element. An enhancer canincrease the efficiency with which a particular gene is transcribed intomRNA irrespective of the distance or orientation of the enhancerrelative to the start site of transcription.

The term “cloning vector” refers to a DNA molecule, such as a plasmid,cosmid, phagemid, or bacteriophage, which has the capability ofreplicating autonomously in a host cell and which is used to transformcells for gene manipulation. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites at whichforeign DNA sequences may be inserted in a determinable fashion withoutloss of an essential biological function of the vector, as well as amarker gene, which is suitable for use in the identification andselection of cells transformed with the cloning vector. Marker genestypically include genes that provide tetracycline resistance orampicillin resistance.

The term “vector” refers to a DNA molecule comprising a clonedstructural gene encoding a foreign protein, which provides a gene in arecombinant host. Typically in the case of a vector destined forintegration into the host genome, the cloned gene is placed or operablylinked to certain upstream and downstream sequences homologous oridentical t the host genetic sequence

The term “recombinant host” refers to a host that may be any prokaryoticor eukaryotic cell, which contains either a cloning vector or expressionvector. This term is also meant to include those prokaryotic oreukaryotic cells that have been genetically engineered to contain thecloned gene(s) in the chromosome or genome of the host cell. Forexamples of suitable hosts, see Sambrook et al., 1989

The terms “express,” “expressing,” “expressed” and “expression” refer toexpression of a gene product (e.g., a biosynthetic enzyme of a gene of apathway or reaction defined and described in this application) at alevel that the resulting enzyme activity of this protein encoded for orthe pathway or reaction that it refers to allows metabolic flux throughthis pathway or reaction in the organism in which this gene/pathway isexpressed in. The expression can be done by genetic alteration of themicroorganism that is used as a starting organism. In some embodiments,a microorganism can be genetically In some embodiments, a microorganismcan be genetically altered (e.g., genetically engineered) to express agene product at an increased level relative to that produced by thestarting microorganism or in a comparable microorganism which has notbeen altered. Genetic alteration includes, but is not limited to,altering or modifying regulatory sequences or sites associated withexpression of a particular gene (e.g. by adding strong promoters,inducible promoters or multiple promoters or by removing regulatorysequences such that expression is constitutive), modifying thechromosomal location of a particular gene, altering nucleic acidsequences adjacent to a particular gene such as a ribosome binding siteor transcription terminator, increasing the copy number of a particulargene, modifying proteins (e.g., regulatory proteins, suppressors,enhancers, transcriptional activators and the like) involved intranscription of a particular gene and/or translation of a particulargene product, or any other conventional means of deregulating expressionof a particular gene using routine in the art (including but not limitedto use of antisense nucleic acid molecules, for example, to blockexpression of repressor proteins).

A microorganism can be physically or environmentally “altered” or“modified” to express a gene product at an increased or lower levelrelative to level of expression of the gene product by the startingmicroorganism. For example, a microorganism can be treated with orcultured in the presence of an agent (chemical or genetic) known orsuspected to increase or decrease the transcription and/or translationof a particular gene and/or translation of a particular gene productsuch that transcription and/or translation are increased or decreased.Alternatively, a microorganism can be cultured at a temperature selectedto increase or decrease transcription and/or translation of a particulargene and/or translation of a particular gene product such thattranscription and/or translation are increased or decreased.“Genetically modified” refers to a microorganism altered in the abovesense by means of genetic engineering techniques available in the art,as for example transformation, mutation, homologous recombination.

The terms “deregulate”, “deregulated” and “deregulation” refer toalteration or modification of at least one gene in a microorganism,wherein the alteration or modification results in increasing efficiencyof SA in the microorganism relative to SA production in absence of thealteration or modification. In some embodiments, a gene that is alteredor modified encodes an enzyme in a biosynthetic pathway or a transportprotein, such that the level or activity of the biosynthetic enzyme inthe microorganism is altered or modified or that the transportspecificity or efficiency is altered or modified. In some embodiments,at least one gene that encodes an enzyme in a biosynthetic pathway isaltered or modified such that the level or activity of the enzyme isenhanced or increased relative to the level in presence of the unalteredor wild type gene. Deregulation also includes altering the coding regionof one or more genes to yield, for example, an enzyme that is feedbackresistant or has a higher or lower specific activity. Also, deregulationfurther encompasses genetic alteration of genes encoding transcriptionalfactors (e.g., activators, repressors), which regulate expression ofgenes coding for enzymes or transport proteins. More specifically,deregulation may result in “decreased” enzyme activity, (wherein theresulting enzyme activity is less than 100% of enzyme activity asobserved in the non-deregulated state is “switched-off”, i.e. reversiblyor irreversibly, no longer present or at least no longer detectable by aconventional analytical took a, like an enzyme activity assay.

The term “capable of utilizing” refers to the ability of a microorganismof the invention to convert a substrate, as for example glycerol into atleast one structurally and/or sterically different chemical product.

An“enzyme activity involved in or associated with the fermentativeconversion of glycerol to succinate” means any catalytic or regulatoryactivity of an enzyme which influences the conversion of glycerol intosuccinate and or by-products, as may be determined by anyone of the setof parameters as defined herein below.

The different yield parameters as described herein (“Yield” or YP/S;“Specific Productivity Yield”; or Space-Time-Yield (STY)) are well knownin the art and are determined as described for example by Song and Lee,2006.

“Yield” and “YP/S” (each expressed in mass of product produced/mass ofmaterial consumed) are herein used as synonyms.

The specific productivity-yield describes the amount of a product, likeSA, that is produced per h and L fermentation broth per g of drybiomass. The amount of dry cell weight stated as DCW describes thequantity of biologically active microorganism in a biochemical reaction.The value is given as g product per g DCW per h (i.e. g/gDCW⁻¹ h⁻¹).

The term “fermentative production” or “fermentation” refers to theability of a microorganism (assisted by enzyme activity contained in orgenerated by said microorganism) to produce a chemical compound in cellculture utilizing at least one carbon source added to the incubation.

The term “fermentation broth” is understood to mean an aqueous solutionwhich is based on a fermentative process and has not been worked up orhas been worked up, for example, as described herein.

b) General Definition for Different Microorganisms

The “bacterial cell” or “bacterial strain” referred to in accordancewith the present invention is selected from the family ofEnterobacteriaceae, Pasteurellaceae, IBacilli or Actinobacteria.

“Enterobacteriaceae” represent a large family of bacteria, includingmany of the more familiar bacteria, such as Salmonella and Escherichiacoli. They belong to the Proteobacteria, and they are given their ownorder (Enterobacteriales). Members of the Enterobacteriaceae arerod-shaped. Like other Proteobacteria they have Gram-negative stains,and they are facultative anaerobes, fermenting sugars to produce lacticacid and various other end products such as succinic acid. Most alsoreduce nitrate to nitrite. Unlike most similar bacteria,Enterobacteriaceae generally lack cytochrome C oxidase. Most have manyflagella used to move about, but a few genera are non-motile. They arenon-spore forming, and mostly they are catalase-positive. Many membersof this family are a normal part of the gut flora found in theintestines of humans and other animals, while others are found in wateror soil, or are parasites on a variety of different animals and plants.Escherichia coli, better known as E. coli, is one of the most importantmodel organisms, and its genetics and biochemistry have been closelystudied. Most members of Enterobacteriaceae have peritrichous Type Ifimbriae involved in the adhesion of the bacterial cells to their hosts.Examples for the enterobacteriaceae are E. coli, Proteus, Salmonella,Klebsiella,

“Pasteurellaceae” comprise a large and diverse family of Gram-negativeProteobacteria with members ranging from bacteria such as Haemophilusinfluenzae to commensals of the animal and human mucosa. Most memberslive as commensals on mucosal surfaces of birds and mammals, especiallyin the upper respiratory tract. Pasteurellaceae are typicallyrod-shaped, and are a notable group of facultative anaerobes. They canbe distinguished from the related Enterobacteriaceae by the presence ofoxidase, and from most other similar bacteria by the absence offlagella. Bacteria in the family Pasteurellaceae have been classifiedinto a number of genera based on metabolic properties and theresequences of the 16S and 23SRNA. A more precise definition ofPasteurellacea can be found in Dousse et al. 2008 and Kuhnert, P. 2008references therein. Many of the Pasteurellaceae containpyruvate-formate-lyase genes and are capable of anaerobically fermentingcarbon sources to organic acids.

The term “Bacilli” refers to a taxonomic class of bacteria. It includestwo orders, Bacillales and Lactobacillales, The bacillus speciesrepresents a large (˜4-8×1.5 im) cylindrical bacteria that can growunder aerobic conditions at 37° C. They are typically nonpathogenic; Thegenus Bacillales cotains the species Alicyclobacillaceae, Bacillaceae,Caryophanaceae, Listeriaceae, Paenibacillaceae, Planococcaceae,Sporolactobacillaceae, Staphylococcaceae, Thermoactinomycetaceae,Turicibacteraceae. Many of the Bacilli contain pyruvate-formate-lyasegenes and are capable of anaerobically fermenting carbon sources toorganic acids.

The term “Actinobacteria” or “Actinomycetes” refers to a group ofGram-positive bacteria with high G+C ratio. They include some of themost common soil life, playing an important role in decomposition oforganic materials. Other Actinobacteria inhabit plants and animals,examples are as Mycobacterium, Corynebacterium, Nocardia, Rhodococcusand Streptomyces. Some Actinobacteria form branching filaments, whichsomewhat resemble the mycelia of the unrelated fungi, among which theywere originally classified under the older name Actinomycetes. Mostmembers are aerobic, but a few can grow under anaerobic conditions.Unlike the Firmicutes, the other main group of Gram-positive bacteria,they have DNA with a high GC-content.

Preferred bacterial strains are of the genus of “Pasteurella”. Thebacteria of the genus Pasteurella are gram-negative and facultativeanaerobic. Pasteurella species are non-motile, pleimorphic and mostoften catalase- and oxidase-positive (Kuhnert and Christensen, 2008,ISBN 978-1-904455-34-9). Preferably, the bacterial cell is a Pasteurellabacterial cell and, more preferably, a Pasteurella strain DD1 cell.

Most preferably, the Pasteurella strain DD1 is the bacterial straindeposited under the Budapest Treaty with DSMZ (Deutsche Sammlung vonMikroorganismen und Zellkulturen, GmbH), Germany, having the depositnumber DSM 18541. This strain has been originally isolated from therumen of a cow of german origin.

Pasteurella bacteria can be isolated from the gastro-intestinal tract ofanimals and, preferably, mammals. The bacterial strain DD1, inparticular, can be isolated from bovine rumen and is capable ofutilizing glycerol (including crude glycerol) as a carbon source.Preferably, the said strain has the ability to produce SA from glycerol(including crude glycerol), in particular, under anaerobic conditions.Moreover, the Pasteurella strain DD1 exhibits at least one of thefollowing additional metabolic characteristics:

a) production of SA from sucrose; in particular, under anaerobicconditions;b) production of succinic acid from maltose; in particular, underanaerobic conditions;c) production of SA from D-fructose; in particular, under anaerobicconditions;d) production of SA from D-galactose; in particular, under anaerobicconditions;e) production of SA from D-mannose; in particular, under anaerobicconditions;f) production of SA from D-glucose; in particular, under anaerobicconditions;g) production of SA from D-xylose; in particular, under anaerobicconditions;h) production of SA from L-arabinose; in particular, under anaerobicconditions;i) no utilization of xylitol, inositol and sorbitol;j) growth both under aerobic and anaerobic conditions;k) growth at initial glucose concentrations of 75 g/L or more;l) ammonia tolerance.

In particular, said strain shows at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11or all of said metabolic characteristics.

Strain DD1 was, analyzed for the capability to co-metabolize asaccharide and the polyol glycerol (PCT/EP2008/006714). It was foundthat DD1 is capable to co-metabolize maltose a d glycerol resulting inbiomass formation, SA formation and simultaneous maltose and glycerolutilization.

c) Preferred Embodiments

A first embodiment of the invention relates to bacterial strain, capableof utilizing glycerol as a carbon source for the fermentative productionof SA wherein said strain is genetically modified so that it comprises aderegulation of its endogenous pyruvate-formate-lyase enzyme activity.In particular, said pyruvate-formate-lyase enzyme activity is decreasedor switched-off.

Said mutated bacterium, containing a pyruvate-formate-lyase with adecreased activity, may be constructed by genetic means as well as byinducing mutations applying methods for mutation well known in the priorart literature (examples and descriptions for the modification ofbacterial genomes can be found in Saier, Milton H Jr 2008, Foster,Patricia L, 2007, Witkin, E M 1969, Eisenstark, A 1971, Walker, G C etal. 1983 and 1984, Botstein, D, and Shortle, D 1985 and referenceswithin, is capable of utilizing mixtures of different carbon sourcessuch as saccharides and glycerol; or utilizing only glycerol. Methods toisolate strains with mutations in the pfl gene can be found in Varenne Set al. 1975, and in Pascal, M et al. 1981.

Preferably said strain has the ability to produce SA from differentcarbon sources (including glycerol), in particular, under anaerobicconditions.

In another embodiment of said strain, at of least one further enzymeactivity involved in or associated with the fermentative conversion ofglycerol to succinate is deregulated.

In particular, said strain is derived from a microorganism selected froma microorganism of the family of Enterobacteriaceae, Pasteurellaceae,Bacilli or Actinobacteria.

In particular, said strain is derived from a microorganism of the familyof Pasteurellaceae, having a 16S rDNA of SEQ ID NO: 1; or a sequence,which shows a sequence homology of at least 96, 97, 98, 99 or 99.9%;and/or having a 23S rDNA of SEQ ID NO: 2; or a sequence, which shows asequence homology of at least 95, 96, 97, 98, 99 or 99.9%. In oneembodiment of the present invention the bacterial strain is derived froma microorganism of the family of Pasteurellaceae and belongs to thespecies Basfia succiniciproducens. The species Basfia succiniciproducensis defined by Kuhnert et al., 2010 incorporated herein by reference.

The bacterial strain of the present invention additionally shows atleast one of the following additional metabolic characteristics:

-   a) production of succinic acid from sucrose;-   b) production of succinic acid from maltose-   c) production of succinic acid from maltodextrin-   d) production of succinic acid from D-fructose;-   e) production of succinic acid from D-galactose;-   f) production of succinic acid from D-mannose;-   g) production of succinic acid from D-glucose;-   h) production of succinic acid from D-xylose;-   i) production of succinic acid from L-arabinose;-   j) production of succinic acid from lactose;-   k) production of succinic acid from raffinose;-   l) production of succinic acid from glycerol;-   m) growth at initial glucose concentrations of 75 g/l or more-   n) growth at initial glycerol concentrations of 70 g/l or more.

as for example a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13or all of said features, with feature I) (glycerol->SA) as a mandatoryconstituent of each of said combinations.

In a further embodiment the strain of the invention is convertingsucrose, maltose, D-fructose, D-glucose, D-xylose, L-arabinose,D-galactose, lactose, D-mannose, raffinose and/or glycerol to succinicacid with a yield coefficient YP/S of at least 0.5 g/g, preferably up toabout 1.28 g/g; as for example a yield coefficient YP/S of at least 0.6g/g, of at least 0.7 g/g, of at least 0.75 g/g, of at least 0.8 g/g, ofat least 0.85 g/g, of at least 0.9 g/g, of at least 0.95 g/g, of atleast 1.0 g/g, of at least 1.05 g/g, of at least 1.07 g/g, of at least1.09 g/g of at least 1.10 g/g, of at least 1.11 g/g, of at least 1.22g/g, or of at least 1.24 g/g

In a further embodiment the strain of the invention shows at least oneof the following characteristics

-   a) converting at least 25 g/L of glycerol to at least 25.1 succinic    acid, with a yield coefficient YP/S of at least 1.01 g/g;-   b) converting at least one carbon source selected from sucrose,    maltose, maltodextrin, D-fructose, D-glucose, D-xylose, L-arabinose,    D-galactose, lactose, D-mannose, raffinose, and/or glycerol to    succinic acid with a specific productivity yield of at least 0.58 g    g DCW⁻¹ h⁻¹ succinic acid;-   c) converting a at least one carbon source selected from sucrose,    maltose, D-fructose, D-glucose, D-xylose, L-arabinose, D-galactose,    lactose, D-mannose, and/or glycerol to succinic add with a space    time yield for succinic acid of at least 2.2 g/(L h) succinic acid;-   d) converting at least 25 g/L of at least one carbon source selected    from sucrose, maltose, D-fructose, D-glucose, D-xylose, L-arabinose,    D-galactose, lactose, D-mannose, and/or glycerol to succinic acid    with a space-time-yield for succinic acid of at least 2.2 g/(L h);-   e) converting at least one carbon source selected from sucrose,    maltose, maltodextrin, D-fructose, D-glucose, D-xylose, L-arabinose,    D-galactose, lactose, D-mannose, raffinose, and/or glycerol to    succinic acid with a specific productivity yield of at least 0.58 g    gDCW⁻¹ h⁻¹ succinic acid and a space-time-yield for succinic acid of    at least 2.2 g/(L h).

According to still another embodiment the bacterial strain of theinvention is converting at least 28 g/L of glycerol to at least 28.1 g/LSA, with a yield coefficient YP/S of at least 1.0 g/g, or of >1.0 g/g,or of >1.05 g/g, or of >1.1 g/g, or of >1.15 g/g, or of >1.20 g/g, orof >1.22 g/g, or of >1.24 g/g, up to about 1.28 g/g. For example, 28 g/Lof glycerol may be converted to up to about 40 or up to about 35 g/L SA.

According to still another embodiment the bacterial strain of theinvention is converting at least one carbon source selected fromsucrose, maltose, raffinose, maltodextrin, D-fructose, D-glucose,D-xylose, L-arabinose, D-galactose, D-mannose, and/or glycerol to SAwith a specific productivity yield of at least 0.6 g g DCW⁻¹ h⁻¹ SA, orof at least of at least 0.65, of at least 0.7 g g DCW⁻¹ h⁻¹, of at least0.75 g gDCW⁻¹ h⁻¹, or of at least 0.77 g gDCW⁻¹ SA.

According to still another embodiment the bacterial strain of theinvention is converting at least one carbon source selected fromsucrose, maltose, raffinose, maltodextrin, D-fructose, D-glucose,D-xylose, L-arabinose, D-galactose, D-mannose, and/or glycerol to SAwith a space time yield for SA of at least 2.2 g/(L h) or of at least2.5, at least 2.75, at least 3, at least 3.25, at least 3.5 or at least3.7 g/(L*h) SA.

According to still another embodiment the bacterial strain of theinvention is converting at least 28 g/L of at least one carbon sourceselected from sucrose, maltose, raffinose, maltodextrin, D-fructose,D-glucose, D-xylose, L-arabinose, D-galactose, D-mannose, and/orglycerol to SA with a space-time-yield for SA of at least 2.2 g/(L h),or of at least 2.5, at least 2.75, at least 3, at least 3.25, at least3.5 or at least 3.7 g/(L*h).

According to another embodiment the bacterial strain of the invention isconverting at least one carbon source selected from sucrose, maltose,raffinose, maltodextrin D-fructose, D-glucose, D-xylose, L-arabinose,D-galactose, D-mannose, and/or glycerol to SA with a specificproductivity yield of at least 0.6 g gDCW⁻¹ h⁻¹ or of at least of atleast 0.65 or of at least 0.7 g gDCW⁻¹ h⁻¹ SA, or of at least 0.77 ggDCW⁻¹ h⁻¹ SA, and a space-time-yield for SA of at least 2.2 g/(L h), orof at least 2.5, at least 2.75, at least 3, at least 3.25, at least 3.5or at least 3.7 g/(L*h).

Preferably said strain of the invention may be derived from strain DD1as deposited with DSMZ having the deposit number DSM 18541 or may be orderived from a variant or mutant strain of DD1 having the ability toproduce succinic acid.

Particular strains of the invention are producing succinic acid (SA) andside products (SSP) in an SA/SSP proportion of >10:1, or >12.5:1,or >15:1, or >17.5,1, or >20:1, or >25:1, or >30:1, or >33:1, whereinSSP represents the sum of side products lactic acid (LA), formic acid(FA), acetic acid (AA), and malic acid (MA), each amount being expressedin g/L.

Further particular strains are producing succinic acid (SA) and the sideproduct acetic acid (AA) in an SA/AA proportion of >10:1, or >12.5:1or >15:1, or >17.5:1, or, >20:1, or >25:1, or >30:1, or >40:1 or >50:1,or >75:1, or >90:1, each amount being expressed in g/L.

Further particular strains are producing succinic acid (SA) and the sideproduct formic acid (FA) in an SA/FA proportion of >90:1, or >100:1,each amount being expressed in g/L.

Another embodiment of the invention relates to a process for thefermentative production of an organic acid or a salt or derivativethereof, which process comprises the steps of:

-   a) incubating a bacterial strain as defined in one of the preceding    claims in a medium containing an assimilable carbon source and    cultivating said strain under conditions favouring the formation of    the desired organic acid; and-   b) obtaining said organic acid, in particular SA, or salt or    derivative thereof from the medium.

According to a particular process the fermentation is performed at atemperature in the range of about 10 to 60° C., as for example 20 to 50°C., 30 to 45° C., or 25 to 35° C., and at a pH of 5.0 to 9.0, as forexample 5.5 to o 8, or 6 t 7, and in the presence of carbon dioxide. ThepH may be controlled by the addition of NH₄HCO₃, (NH₄)₂CO₃, NaOH,Na₂CO₃, NaHCO₃, KOH, K₂CO₃, KHCO₃, Mg(OH)₂, MgCO₃, MgH(CO₃)₂, Ca(OH)₂,CaCO₃, Ca(HCO₃)₂, CaO, CH₆N₂O₂, C₂H₇N and/or mixtures thereof.

In particular, said assimilable carbon source is selected from glycerol,sucrose, maltose, maltodextrin, D-fructose, D-galactose, D-mannose,lactose, D-glucose, D-xylose, L-arabinose, raffinose decompositionproducts of starch, cellulose, hemicelluloses and lignocellulose; andmixtures thereof.

In particular, said carbon source is glycerol or a mixture of glyceroland at least one further carbon source selected from sucrose, maltose,D-fructose, D-galactose, lactose, D-mannose, D-glucose, D-xylose,raffinose and L-arabinose.

According to a particular embodiment of said process, the concentrationof the assimilable carbon source is adjusted to a value in a range of 5to 80 g/l, as for example 10 to 60.

The present invention further provides a process for the fermentativeproduction of succinic acid or a salt or derivative thereof, whichprocess comprises the steps of:

-   a) incubating a bacterial strain in a medium containing at least one    assimilatable carbon source and cultivating said strain under    conditions favoring the formation of the desired organic acid;-   b) obtaining said organic acid or salt or derivative thereof from    the medium;    -   additionally characterized by at least one of the following        features:-   c) conversion of at least 25 g/L of glycerol to at least 25.1 g/L    succinic acid, with a yield coefficient YP/S of at least 1.0 g/g-   d) conversion of at least one carbon source selected from sucrose,    maltose, D-fructose, D-glucose, D-xylose, L-arabinose, D-galactose,    D-mannose, raffinose, and/or glycerol to succinic acid with a    specific productivity yield of at least 0.58 g gDCW⁻¹ h⁻¹ succinic    acid;-   e) conversion of at least one carbon source selected from sucrose,    maltose, D-fructose, D-glucose, D-xylose, L-arabinose, D-galactose,    lactose, D-mannose, raffinose and/or glycerol to succinic acid with    a space time yield for succinic acid of at least 2.2 g/(L h)    succinic acid;-   f) conversion of at least 25 g/L of at least one carbon source    selected from sucrose, maltose, D-fructose, D-glucose, D-xylose,    L-arabinose, D-galactose, lactose, D-mannose, raffinose, and/or    glycerol to succinic acid with a space-time-yield for succinic acid    of at least 2.2 g/(L h);-   g) conversion of at least one carbon source selected from sucrose,    maltose, D-fructose, D-glucose, D-xylose, L-arabinose, D-galactose,    lactose, D-mannose, raffinose, and/or glycerol to succinic acid with    a specific productivity yield of at least 0.6 g gDCW⁻¹ h⁻¹ succinic    acid and a space-time-yield for succinic acid of at least 2.2 g/(L    h;-   h) production of succinic acid (SA) and side products (SSP) in an    SA/SSP proportion of >10:1, or >12.5:1, or >15:1, or >17.5,1,    or >20:1, or >25:1, or >30:1, or >33:1, wherein SSP represents the    sum of side products lactic acid (LA), formic acid (FA), acetic acid    (AA), and malic acid (MA), each amount being expressed in g/L;-   i) production of succinic acid (SA) and the side product acetic acid    (AA) in an SA/AA proportion of >10:1, or >12.5:1, or >15:1,    or >17.5:1, or >20:1, or >25:1, or >30:1, or 50:1, or >75:1,    or >90:1, each amount being expressed in g/L.

According to a particular embodiment of said process said bacterialstrain is a genetically modified strain as defined above.

The processes of the invention may be performed discontinuously orcontinuously. The course of the acid production may be monitored byconventional means, as for example HPLC or GC analysis.

Preferably SA is produced under anaerobic conditions. Anaerobicconditions may be established by means of conventional techniques, asfor example by degassing the constituents of the reaction medium andmaintaining anaerobic conditions by introducing carbon dioxide ornitrogen or mixtures thereof and optionally hydrogen at a flow rate of,for example, 0.1 to 1 or 0.2 to 0.5 vvm.

Aerobic conditions may be established by means of conventionaltechniques, as for example by introducing air or oxygen at a flow rateof, for example, 0.1 to 1 or 0.2 to 0.5 vvm.

If appropriate a slight over pressure of 0.1 to 1.5 bar may be appliedaccording to the invention.

In another embodiment the invention provides a process for theproduction of succinic acid and/or succinic acid ammonium salts whichmethod comprises the fermentative production of succinic acid as definedabove and additionally controlling the pH with a base ammonia or anaqueous solution thereof, or with NH₄HCO₃, (NH₄)₂CO₃, NaOH, Na₂CO₃,NaHCO₃, KOH, K₂CO₃, KHCO₃, Mg(OH)₂, MgCO₃, MgH(CO₃)₂, Ca(OH)₂, CaCO₃,Ca(HCO₃)₂, CaO, CH₆N₂O₂, C₂H₇N and mixtures thereof. Generally, thephysical condition of the base can either be an aqueous solution,aqueous suspension, gaseous or solid.

In one embodiment the organic acid, in particular succinic acid, and/orsalts thereof are produced by one of the above or below mentionedmethods and are further isolated and/or purified by the following steps:

-   -   filtration and/or centrifugation,    -   cation exchange chromatography and/or    -   crystallization.

-   Preferably the organic acid and/or salts thereof are further    isolated and/or purified by the following steps:    -   filtration, followed by    -   cation exchange chromatography, followed by    -   crystallization.

The filtration may be used to separate the bacterial cells from thesuccinic acid containing liquid. The filtration may be a diafiltrationcrossflow-filtration and/or ultrafiltration.

The material used for cation exchange chromatography may be a strongacid cation exchange resin. A strong acid cation exchange resin carriesfor example sulfonic acid groups. In particular, the material used forcation exchange chromatography may be a styrol-divinylbenzol-copolymerisate carrying sulfonic acid groups in the H⁺-form.H⁺-form means that the sulfonic acid groups are present in theacid-form. Preferably, the average particle size of the cation exchangechromatography resin is 0.3 to 1.5, more preferably 0.55 to 0.75 mmand/or the bulk density is 700 to 800 g/l. The cation exchangechromatography resin may be macroporous. Makroporous means thatpreferably the average pore diameter of the cation exchange resin isfrom 20 to 120 nm, preferably from 20 to 100 nm and more preferably from20 to 40 nm. The particle distribution is preferably monodispers.Preferably, the total capacity of the cation exchange chromatographymaterial is 0.5 to 2.0, more preferably 0.8 to 1.7, more preferably 1.0to 1.5, more preferably 1.4 to 1.9 min eq./l., x eq./l means that 1 lcation exchange resin carries x mol sulfonic acid groups. Accordinglyeq./l is calculated with respect to a single charged molecule. Thesuccinic acid salt to be purified may be a Na, K, Ca, Mg and/or ammoniumsalt. For example the strong acid cation exchange resin may be TypeLewatit Monoplus SP 112 from Lanxess.

Preferably, the cation exchange chromatography is performed at atemperature from 20 to 60° C., more preferably from 46 to 60° C.

Further preferred methods of producing SA are described below:

Method 1:

In another embodiment the present invention provides a process for thefermentative production of SA or a salt or derivative thereof, whichprocess comprises the steps of:

-   a. incubating a bacterial strain in a medium containing at least one    assimilatable carbon source and cultivating said strain under    conditions favoring the formation of the desired organic acid;-   b. obtaining said organic acid or salt or derivative thereof from    the medium;-    and which process is additionally characterized by conversion of at    least 50 g/L of glycerol to at least 50 g/L SA, with a yield    coefficient YP/S of at least 1.0 g/g, or of >1.0 g/g, or of >1.05    g/g, or of >1.1 g/g, or of >1.15 g/g, or of >1.20 g/g, or of >1.22    g/g, or of >1.24 g/g; up to about 1.28 g/g; as for example a yield    coefficient YP/S of at least 0.6 g/g, of at least 0.7 g/g, of at    least 0.75 g/g, of at least 0.8 g/g, of at least 0.85 g/g, of at    least 0.9 g/g, of at least 0.95 g/g, of at least 1.0 g/g, of at    least 1.05 g/g, of at least 1.1 g/g, of at least 1.15 g/g, of at    least 1.20 g/g, of at least 1.22 g/g, or of at least 1.24 g/g. For    example, 50 g/L of glycerol may be converted to up to about 65 or up    to 62.5 g/L SA or up to 60 g/L SA.

Method 2:

In another embodiment the present invention provides a process for thefermentative production of SA or a salt or derivative thereof, whichprocess comprises the steps of:

-   a. incubating a bacterial strain with a pyruvate-formate-lyase    enzyme with decreased activity in a medium containing at least one    assimilatable carbon source and cultivating said strain under    conditions favoring the formation of the desired organic acid;-   b. obtaining said organic acid or salt or derivative thereof from    the medium; and which process is additionally characterized by-    conversion of a carbon source selected from sucrose, maltose,    maltodextrin, raffinose, D-fructose, D-glucose. D-xylose,    L-arabinose, D-galactose, D-mannose, and/or glycerol to SA with a    specific productivity yield of at least 0.42. g gDCW⁻¹ h⁻¹ SA or of    at least of at least 0.45 or of at least 0.47 g g DCW⁻¹ h⁻¹ SA, or    of at least 0.49 g gDCW⁻¹ h⁻¹ SA.

Method 3:

In another embodiment the present invention provides a process for thefermentative production of SA or a salt or derivative thereof, whichprocess comprises the steps of:

-   a. incubating a bacterial strain with a pyruvate-formate-lyase    enzyme with decreased activity in a medium containing at least one    assimilatable carbon source and cultivating said strain under    conditions favoring the formation of the desired organic acid;-   b. obtaining said organic acid or salt or derivative thereof from    the medium;-    and which process is additionally characterized by conversion of a    carbon source selected from sucrose, maltose, maltodextrin,    raffinose, D-fructose, D-glucose, D-xylose, L-arabinose,    D-galactose, D-mannose, and/or glycerol to SA with a space time    yield for SA of at least 2.22 g/(L h), or of at least 2.5, at least    2.75, at least 2.9, g/(L*h) SA.

Method 4:

In another embodiment the present invention provides a process for thefermentative production of SA or a salt or derivative thereof, whichprocess comprises the steps of:

-   a. incubating a bacterial strain in a medium containing at least one    assimilable carbon source and cultivating said strain under    conditions favoring the formation of the desired organic acid;-   b. obtaining said organic acid or salt or derivative thereof from    the medium;-    and which process is additionally characterized by conversion of at    least 50 g/L of a source selected from sucrose, maltose,    maltodextrin, raffinose, D-fructose, D-glucose, D-xylose,    L-arabinose, D-galactose, D-mannose, and/or glycerol to SA with a    space-time-yield for SA of at least 2.2 g/(L h), or of at least 2.5,    at least 2.75, at least 3, at least 3.25, at least 3.5 or at least    3.7 g/(L*h).

Method 5:

In another embodiment the present invention provides a process for thefermentative production of SA or a salt or derivative thereof, whichprocess comprises the steps of:

-   a. incubating a bacterial strain in a medium containing at least one    assimilatable carbon source and cultivating said strain under    conditions favoring the formation of the desired organic acid;-   b. obtaining said organic acid or salt or derivative thereof from    the medium;-    and which process is additionally characterized by conversion of a    carbon source selected from sucrose, maltose, maltodextrin,    raffinose, D-fructose, D-glucose, D-xylose, L-arabinose,    D-galactose, D-mannose, and/or glycerol to SA with a specific    productivity yield of at least 0.6 g gDCW⁻¹ h⁻¹ SA or of at least of    at least 0.65 or of at least 0.7 g gDCW⁻¹ h⁻¹ SA, or of at least    0.75 g gDCW⁻¹ h⁻¹ SA, or of at least 0.77 g gDCW⁻¹ h⁻¹ SA and a    space-time-yield for SA of at least of at least 2.2 g/(L h), or of    at least 2.5, at least 2.75, at least 3, at least 3.25, at least 3.5    or at least 3.7 g/(L*h).

In another embodiment of the above identified methods 1 to 5 ofproducing SA the carbon source is glycerol or a mixture of glycerol andat least one further carbon source selected from sucrose, maltose,raffinose, maltodextrin, D-fructose, D-galactose, D-mannose, D-glucose,D-xylose, and L-arabinose.

Particularly suitable conditions for producing SA are:

-   -   Carbon source: Glucose, xylose, maltose or maltodextrin,        raffinose and/or glycerol (including crude glycerol)    -   Temperature: 30 to 45° C.    -   pH: 5.5 to 7.0, controlled by a base as described above,        preferably by a HCO₃— source such as Na₂CO₃, NaHCO₃, Mg(HCO₃)₂,        Ca(HCO₃)₂ or, Mg(OH)₂, MgCO₃, Ca(OH)₂, CaCO₃, supplied gas: CO₂        SA and/or SA salts produced may be isolated in conventional        manner by methods known in the art, as for example        crystallization, filtration, electrodialysis, chromatography.        For example, they may be isolated by precipitating as a calcium        succinate product in the fermenter during the fermentation by        using calcium hydroxide, -oxide, -carbonate or hydrogencarbonate        for neutralization and filtration of the precipitate. The        desired SA product is recovered from the precipitated calcium        succinate by acidification of the succinate with sulfuric acid        followed by filtration to remove the calcium sulfate (gypsum) or        which precipitates. The resulting solution may be further        purified by means of ion exchange chromatography in order to        remove undesired residual ions.

In another embodiment the present invention provides a process for theproduction of tetrahydrofuran (THF) and/or 1,4-butanediol (BDO) and/orgamma-butyrolactone (GBL), which comprises

-   b) the fermentative production of succinic acid and/or succinic acid    salts, as defined above, and-   b1) either the direct catalytic hydrogenation of the obtained free    acid to THF and/or BDO and/or GBL or-   b2) the chemical esterification of obtained free succinic acid    and/or succinic acid salts to its corresponding di-loweralkyl ester    and subsequent catalytic hydrogenation of said ester to THF and/or    BDO and/or GBL.

In another embodiment the present invention provides a process for theproduction of pyrrolidones which comprises

-   a) the fermentative production of succinic acid ammonium salts as    defined above, and-   b) the chemical conversion of succinic acid ammonium salts to    pyrrolidones in a manner known per se.

In a particular embodiment of the processes of the said glycerol, whichis used as assimilable carbon source, is crude glycerol, in particularobtained by ester cleavage of triacylglycerides. For example glycerol isa waste product as obtained from the manufacture of bio diesel.

The present invention also relates to the use of a bacterial strain asdefined above for the fermentative production of an organic finechemical, as for example succinic acid or a salt or derivative thereof.

d) Further Particular Embodiments

d1) Genetic Manipulations

According to still another embodiment the bacterial strain of theinvention contains a gene coding for a mutated enzyme of thepyruvate-formate-lyase (pfl) enzyme as its enzymatic activity is definedby the EC number EC 2.3.1.54. For example pfl enzyme activity isnegatively influenced by the mutations in the pflA gene or by affectingthe expressional regulation of the pflA gene. The sequence of the pflAgene and the pflA gene product can be found under the followingaccession numbers GeneID:6268899, YP_(—)001880903: Homologues of thisgene are known under the accession numbers: NCBI-GeneID 945514, 945444,947623, 948454, 3075405, the respective proteins under the accessionnumbers: UniProt: P09373, P75793, P42632, P32674, Q65VK2.

Also within in the scope of this invention are genes coding for thepyruvate-formate-lyase activating enzymes which are defined by the ECnumber EC 1.97.1.4 and are described in Knappe et al. 1990 and 1993 withdecreased or deregulated activity. This can be performed throughintroducing mutations or gene deletions by methods described in thisinvention. Examples for this enzyme which activities can be decreased orwhich coding gene can be mutated or deregulated are encoded by the pflactivating enzyme gene pflA and the yfiD gene, the E. coli K12 geneknown under the accession GeneID: 947068, the gene ybiY, with theaccession number NCBI-GeneID: 945445 and the respective proteinNP_(—)415345 the Mannheimia succiniproducens gene known under theaccession GeneID: AAU37008, the respective proteins under the accessionYP_(—)087593, NP_(—)417074 and YP_(—)087564 as well as the homologues ofthis gene. Described are the accession numbers of the non-mutated genesequences which are subject to mutations or deletions described in thisinvention.

Also in the scope of this invention are strains showing a reducedactivity of the protein arcA eg. Accession: ECK4393 (also known underthe following descriptions: cpxC, fexA, sfrA, msp) or fnr by carryinggenetic mutations for the respective gene, known under the accessionNCBI-GeneID: 948874 for arcA or NCBI-GeneID: 945908 for fnr. Respectiveprotein sequences can be found under the Accession P0A9E5. Similar genesare known for other organisms such as Mannheimia succinicproducensnamely. NCBI-GeneID: 3076294 and the respective protein YP_(—)088696 forarcA and for fnr NCBI-GeneID:3075449 and UniProt: Q65TM6.

Also in the scope of this invention are strains showing a reducedactivity of the lactate dehydrogenase defined by the EC number EC1.1.1.27 and EC 1.1.1.28 coding for enzymes with a specificity ofproducing D-lactic or L-lactic acid or both. Examples are the E. coligenes NCBI-GeneID 946315 and the respective protein NP_(—)415898 or theM succiniproducens gene NCBI-GeneID 3075603 and the respective proteinYP_(—)089271.

According to still another embodiment the bacterial strain of theinvention contains:

(1) a mutated gene coding for a pyruvate-formate-lyase enzyme defined bythe EC nomenclature as EC 2.3.1.54, with decreased activity; and/or (2)a mutated gene coding for the pyruvate-formate-lyase activating enzymedefined by the EC nomenclature as EC 1.97.1.4 with decreased activity;and/or (3) a mutated gene coding for the arcA protein and/or (4) amutated gene coding for a lactate dehydrogenase defined by the ECnomenclature as EC 1.1.1.27 or EC 1.1.1.28 with decreased activity.

A particular method for preparing genetically modified bacterial strainsof the invention is a technique that is also sometimes referred to asthe “Campbell recombination” herein (Leenhouts et al., 1989, Appl EnvMicrobiol 55, 394-400). “Campbell in”, as used herein, refers to thepreparation of a transformant of an original host cell in which anentire circular double stranded DNA molecule (for example a plasmid) hasintegrated into a chromosome by a single homologous recombination event(a cross in event), and that effectively results in the insertion of alinearized version of said circular DNA molecule into a first DNAsequence of the chromosome that is homologous to a first DNA sequence ofthe said circular DNA molecule. “Campbelled in” refers to the linearizedDNA sequence that has been integrated into the chromosome of a “Campbellin” transformant. A “Campbell in” contains a duplication of the firsthomologous DNA sequence, each copy of which includes and surrounds acopy of the homologous recombination crossover point.

“Campbell out,” as used herein, refers to a cell descending from a“Campbell in” transformant, in which a second homologous recombinationevent (a cross out event) has occurred between a second DNA sequencethat is contained on the linearized inserted DNA of the “Campbelled in”DNA, and a second DNA sequence of chromosomal origin, which ishomologous to the second DNA sequence of said linearized insert. Thesecond recombination event results in the deletion (jettisoning) of aportion of the integrated DNA sequence, but, importantly, also resultsin a portion (this can be as little as a single base) of the integrated“Campbelled in” DNA remaining in the chromosome, such that compared tothe original host cell, the “Campbell out” cell contains one or moreintentional changes in the chromosome (for example, a deletion of theDNA sequence, a single base substitution, multiple base substitutions,insertion of a heterologous gene or DNA sequence, insertion of anadditional copy or copies of a homologous gene or a modified homologousgene, or insertion of a DNA sequence comprising more than one of theseaforementioned examples listed above).

A “Campbell out” cell is, preferably, obtained by a counter-selectionagainst a gene that is contained in a portion (the portion that isdesired to be jettisoned) of the “Campbelled in” DNA sequence, forexample the Bacillus subtilis sacB gene, which is lethal when expressedin a cell that is grown in the presence of about 5% to 10% sucrose.Either with or without a counter-selection, a desired “Campbell out”cell can be obtained or identified by screening for the desired cell,using any screenable phenotype, such as, but not limited to, colonymorphology, colony color, presence or absence of antibiotic resistance,presence or absence of a given DNA sequence by polymerase chainreaction, presence or absence of an auxotrophy, presence or absence ofan enzyme, presence or absence of an enzymatic activity such as apyruvate formate lyase activity or a lactate dehydrogenase activity,colony nucleic acid hybridization, antibody screening, etc. The term“Campbell in” and “Campbell out” can also be used as verbs in varioustenses to refer to the method or process described above.

It is understood that the homologous recombination event that leads to a“Campbell in” or “Campbell out” can occur over a range of DNA baseswithin the homologous DNA sequence, and since the homologous sequenceswill be identical to each other for at least part of this range, it isnot usually possible to specify exactly where the crossover eventoccurred. In other words, it is not possible to specify precisely whichsequence was originally from the inserted DNA, and which was originallyfrom the chromosomal DNA. Moreover, the first homologous DNA sequenceand the second homologous DNA sequence are usually separated by a regionof partial non-homology, and it is this region of non-homology thatremains deposited in a chromosome of the “Campbell out” cell.

Preferably, first and second homologous DNA sequence are at least about200 base pairs in length, and can be up to several thousand base pairsin length. However, the procedure can be made to work with shorter orlonger sequences. For example, a length for the first and secondhomologous sequences can range from about 500 to 2000 bases, and theobtaining of a “Campbell out” from a “Campbell in” is facilitated byarranging the first and second homologous sequences to be approximatelythe same length, preferably with a difference of less than 200 basepairs and most preferably with the shorter of the two being at least 70%of the length of the longer in base pairs.

By applying the above method of genetic modification, mutant strains ofa particular SA producer strain (i.e. DD1) were prepared by deleting thegens of endogenous pyruvate-formate-lyase enzyme and/or lactatedehydrogenase enzyme as described in more detail in the followingexamples.

d2) Fermentation Steps:

A fermentation as used according to the present invention can, forexample, be performed in stirred fermentors, bubble columns and loopreactors. A comprehensive overview of the possible method typesincluding stirrer types and geometric designs can be found in “Chmiel:Bioprozesstechnik: Einführung in die Bioverfahrenstechnik, Band 1”. Inthe process of the invention, typical variants available are thefollowing variants known to those skilled in the art or explained, forexample, in “Chmiel, Hammes and Bailey: Biochemical Engineering”, suchas batch, fed-batch, repeated fed-batch or else continuous fermentationwith and without recycling of the biomass. Depending on the productionstrain, sparging with air, oxygen, carbon dioxide, hydrogen, nitrogen orappropriate gas mixtures may be effected in order to achieve good yield(YP/S).

Before the intended chemical conversion in a fermentation broth isperformed in the process according to the invention, the fermentationbroth can be pretreated; for example, the biomass of the broth can beremoved. Processes for removing the biomass are known to those skilledin the art, for example filtration, sedimentation and flotation.Consequently, the biomass can be removed, for example, with centrifuges,separators, decanters, filters or in flotation apparatus. For maximumrecovery of the product of value, washing of the biomass is oftenadvisable, for example in the form of a diafiltration. The selection ofthe method is dependent upon the biomass content in the fermenter brothand the properties of the biomass, and also the interaction of thebiomass with the product of value. In one embodiment, the fermentationbroth can be sterilized or pasteurized.

In a further embodiment, the fermentation broth is concentrated.Depending on the requirement, this concentration can be done batch wiseor continuously. The pressure and temperature range should be selectedsuch that firstly no product damage occurs, and secondly minimal use ofapparatus and energy is necessary. The skillful selection of pressureand temperature levels for a multistage evaporation in particularenables saving of energy.

Stirred tanks, falling-film evaporators, thin-film evaporators,forced-flash circulation evaporators and other evaporator types can beutilized in natural or forced circulation mode.

d3) Esterification of SA and Hydrogenation:

Suitable experimental conditions for performing the chemicalesterification, followed by direct catalytic hydrogenation are wellknown, and for example, described in European Patent application06007118.0 incorporated herewith by reference.

a) Esterification Process:

The esterification process, which may comprise a reactive distillationcan be performed using an apparatus known per se in various designs.

For example an esterification plant, which is operated in continuousmode can be used which comprises a rectification column with anappropriate number of theoretical stages achieved by installation oftrays or packings. The aqueous charge comprising the ammonium salt of SAis fed into the top of the column from a reservoir vessel as soon as asteady-state temperature profile has formed in the column as a result offeeding-in alkanol that is evaporated in the evaporator loop adherent tothe sump of the column. The reaction forms a countercurrent flow ofdescending, ammonium salt-containing liquid and condensate, andascending, alkanol-containing vapor phase. To catalyze theesterification reaction, a homogeneous catalyst may be added to theammonium salt initial charge. Alternatively, heterogeneous catalysts maybe provided in the column internals. The carboxylic ester formed isliquid under the process conditions and passes via the lower end of thecolumn into the sump of the distillation column and is continuouslywithdrawn from the sump. Gaseous components, for example azeotropicmixtures comprising alkanol-water and/or ammonia, are removed from thereaction column and hence from the reaction equilibrium at the top ofthe column.

Further modifications of the above-described specific embodiments can beimplemented by the person skilled in the art without unacceptableeffort.

Suitable process parameter ranges for the esterification processaccording to the invention can be determined easily by the personskilled in the art depending on the configuration of the apparatus used,for example type of column internals used, type and amount of thereactants, type and amount of the catalyst used if appropriate. Forinstance, without being restrictive thereto, individual parameters maybe set within the following parameter ranges:

Column temperature: 0-300° C., in particular 40-250° C., or 70-200° C.

Pressure: from 0.1 to 6 bar, in particular standard pressure

Residence time: a few seconds (for example from 1 to 60) up to days (forexample from 1 to 5),

in particular from a few minutes (for example from 1 to 60) to a fewhours (for example from 1 to 15), more preferably from a few minutes(for example from 5 to 20) to 2 h.

b) Hydrogenation Process

The SA esters or SA as prepared in accordance with the invention per seare hydrogenated in a manner known per se using processes, apparatus andassistants, such as catalysts, familiar to the person skilled in theart.

In particular, a continuous or batchwise gas phase hydrogenation iscarried out in the presence of a heterogeneous catalyst suitable for theester hydrogenation. The optimal process parameters can be establishedby the person skilled in the art for the particular ester withoutunacceptable effort. For example, the reaction temperature is in therange from about 100 to about 300° C., preferably in the range fromabout 200 to 280° C., and the pressure is from about 5 to 100 bar, forexample from 10 to 50 bar. The molar ratio of reactant to hydrogen isset within the range from about 1:100 to about 1:2000, for example from1:800 to 1:1500.

Catalysts usable for the hydrogenation reaction are known to the personskilled in the art. For example, various copper catalysts may be used.The prior art describes, for example, the use of reduced copper chromitecatalysts which are obtainable under the name 85/1 from Davy ProcessTechnology Ltd., England. However, catalysts particularly suitable inaccordance with the invention are supported copper oxide catalysts, thecopper oxide being applied to alumina or silica support materials. Theexamples of the hydrogenation of succinic esters to BDO(1,4-Butanediol)/GBL (gamma-butyrlactone)/THF with copper catalysts arealso described in the following thesis: Schlander, January, February2000, University of Karlsruhe, “Gasphasenhydrierung vonMaleinsäuredimethylester zu 1,4-Butandiol, gamma-Butyrolacton undTetrahydrofuran an Kupfer-Katalysatoren”.

The present invention will be described in greater detail by means ofthe following examples. The following examples are for illustrativepurposes and are not intended to limit the scope of the invention.

EXAMPLES Genetic Modification and Cultivation Example 1 General Methodfor the Transformation of DD1

TABLE 1 Nomenclature of the DD1 wildtype and mutants referred to in theexamples. Strain Description LU13843 Wildtype DD1 (deposit DSM18541)LU15348 DD1 Δ pfl LU15050 DD1 Δ ldh LU15224 DD1 Δ pfl Δ ldh

Pasteurella strain LU13843 (wild type DD1) was transformed with DNA byelectroporation using the following protocol:

For preparing a pre-culture LU 13843 was inoculated from a freshly grownBHI-Agar plate into 40 ml BHI (brain heart infusion, Difco) in 100 mlshake flask. Incubation was performed over night at 30° C.; 200 rpm.

For preparing the main-culture 50 ml BHI were placed in a 100 ml shakeflask and inoculated to a final OD (610 nm) of 0.4 with the preculture.Incubation was performed for approximately 1.5 h at 30° C., 200 rpm. Thecells were harvested at an OD of approximately 1.3, pellet were washedonce with 10% cold glycerol at 4° C. and resuspended in 1.7 ml 10%glycerol (4° C.).

100 μl of competent cells were the mixed with 5-10 μg DNA (10-20 μl) andkept on ice for 2 min in an electroporation cuvette with a width of 0.2cm. Electroporation under the following conditions: 800 Ω; 25 μF; 2 kV(Gene Pulser, Bio-Rad). 1 ml of BHI was added immediately afterelectroporation. and an incubation was performed for 2 h at 30° C.

Cells were plated on BHI with 5 mg/L chloramphenicol and incubated for2-5 d at 30° C. until the colonies of the transformants were visible.Clones were isolated and restreaked onto BHI with 5 mg/l chloramphenicoluntil purity of clones was obtained.

Example 2 Generation of Deletion Constructs

Mutation/deletion plasmids were constructed based on the vector pSacB(SEQ ID NO 3). FIG. 1 shows a schematic map of plasmid pSacB. 5′- and3′-flanking regions of the chromosomal fragment, which should be deletedwere amplified by PCR from chromosomal DNA of LU 13843 and introducedinto said vector using standard techniques. Normally, at least 80% ofthe ORF were targeted for a deletion. In such a way, the deletionplasmids for the pyruvate-formate-lyase pfl, pSacB (Δpfl) (SEQ ID NO 4),and the lactate dehydrogenase ldhA, pSacB (Δ ldhA) (SEQ ID NO 5) wereconstructed. FIGS. 2 and 3 show schematic maps of plasmid pSacB (Δpfl)and pSacB (ΔldhA).

In the plasmid sequence of pSacB (SEQ ID NO:3) the sacB gene iscontained from bases 5169-6590. The chloramphenicol gene is containedfrom base 526-984. The sacB promotor is contained from bases 3802-4264.The chloramphenicol gene is contained from base 526-984. The origin ofreplication for E. coli (ori EC) is contained from base 1477-2337.

In the plasmid sequence of pSacB delta pfl (SEQ ID NO:4) the 3′ flankingregion of the pfl gene, which is homologous to the genome of DD1, iscontained from bases 65-1533, while the 5′ flanking region of the pflgene which is homologous to the genome of DD1 is contained from bases1534-2956. The sacB gene is contained from bases 5256-6677. The sacBpromoter is contained from bases 6678-7140. The chloramphenicol gene iscontained from base 3402-3860. The origin of replication for E. coli(ori EC) is contained from base 4353-5213.

In the plasmid pSacB delta ldh (SEQ ID NO:5) the 5′ flanking region ofthe ldh gene, which is homologous to the genome of DD1, is containedfrom bases 2850-1519, while the 3′ flanking region of the ldh gene,which is homologous to the genome of DD1, is contained from bases1518-63. The sacB gene is contained from bases 5169-6590. The sacBpromoter is contained from bases 6591-7053. The chloramphenicol gene iscontained from base 3315-3773. The origin of replication for E. coli(ori EC) is contained from base 4266-5126.

Example 3 Generation of Improved Succinate Producing Strains

a) LU 13843 was transformed as described above with the pSacB (Δpfl) and“Campbelled in” to yield a “Campbell in” strain. Transformation andintegration into the genome of LU 13843 was confirmed by PCR yieldingbands for the integrational event of the plasmid into the genome of LU13843.

The “Campbell in” strain was then “Campbelled out” using agar platescontaining sucrose as a counter selection medium, selecting for the loss(of function) of the sacB gene. Therefore, the “Campbell in” strainswere incubated in 25-35 ml of non selective medium (BHI containing noantibiotic) at 37° C., 220 rpm over night. The overnight culture wasthen streaked onto freshly prepared BHI containing sucrose plates (10%,no antibiotics) and incubated overnight at 37° C. (“first sucrosetransfer”). Single colony obtained from first transfer were againstreaked onto freshly prepared BHI containing sucrose plates (10%) andincubated overnight at 37° C. (“second sucrose transfer”). Thisprocedure was repeated until a minimal completion of five transfers(“third, forth, fifth sucrose transfer”) in sucrose. The term “first tofifth sucrose transfer” refers to the transfer of a strain afterchromosomal integration of a vector containing a sacB levansucrase geneonto sucrose and growth medium containing agar plates for the purpose ofselecting for strains with the loss of the sacB gene and the surroundingplasmid sequences. Single colony from the fifth transfer plates wereinoculated onto 25-35 ml of non selective medium (BHI containing noantibiotic) and incubated at 37° C., 220 rpm over night. The overnightculture was serially diluted and plated onto BHI plates to obtainisolated single colonies.

The “Campbelled out” strains containing, the mutation/deletion of thepfl gene were confirmed by chloramphenicol sensitivity. Themutation/deletion mutants among these strains were identified andconfirmed by PCR analysis. This led to the pfl mutation/deletion mutantDD1 delta pfl LU 15348.

b) LU15348 was transformed with pSacB (Δ ldh) as described above and“Campbelled in” to yield a “Campbell in” strain. Transformation andintegration was confirmed by PCR. The “Campbell in” strain was then“Campbelled out” as described previously. The deletion mutants amongthese strains were identified and confirmed by PCR analysis. This led tothe pfl ldhA double deletion mutant LU15224.

c) LU13843 was transformed with pSacB (Δ ldh) as described above and“Campbelled in” to yield a “Campbell in” strain. Transformation andintegration was confirmed by PCR. The “Campbell in” strain was then“Campbelled out” as described previously. The deletion mutants amongthese strains were identified and confirmed by PCR analysis. This led tothe ldhA deletion mutant LU15050.

Example 4 Cell Bank Preparation 1. Media Preparation

Composition of the cultivation media is described in table 2.

TABLE 2 Composition of solid and liquid media for the preparation ofcell banks. Concentration Concentration of Compound [g/l] stock solution[g/l] Glucose varying^(a) 650 Bacto yeast extrakt (Becton 5 — Dickinson)Bacto peptone (Becton Dickinson) 5 — (NH4)₂SO₄ 1 500 CaCl₂*2H₂O 0.2 20MgCl₂*6H₂O 0.2 20 NaCl 1 100 K₂HPO₄ 3 500 MgCO₃ Varying^(b) — Bacto-Agar(for solid media only) 12 ^(a)Glucose concentrations were 15 g/l (inplates) and 20 or 50 g/l (in liquid media). ^(b)MgCO₃ (Riedel-de Haen,product number: 13117 by Sigma-Aldrich Laborchemikalien GmbH)concentrations were 5 g/l (in plates) and 0 or 30 g/l (in liquid media).

5 g yeast extract, 5 g peptone, MgCO₃ and (for solid media) 12 gBacto-Agar were mixed in 900 ml distilled water and autoclaved (20 min).After cooling down to about 65° C. the missing components were added assterile stock solutions. Glucose, ammonium sulfate and K₂HPO₄ were allseparately autoclaved. Ca-, Mg- and Na-chlorides were autoclavedtogether.

2. MCB Preparation

The master cell bank (MCB) for the inoculation of the individualexperiments was performed as followed. Two agar plates were freshlyinoculated with the desired strain and incubated at 37° C. in ananaerobic jar (Anaerocult A, Merck) over night. The biomass was takenoff the plates and resuspended in the MgCO₃-free liquid medium with 20g/l glucose to adjust OD₆₀₀≈1.0. Inoculation was performed with 0.5 mlof this cell suspension. Cultivations were performed in 100 ml-serumbottles with gas tight butyl rubber stoppers (Ochs GmbH,Bovenden/Lenglern, Germany) containing 50 ml of the liquid medium with20 g/l glucose and 30 g/l MgCO₃ and a CO₂— atmosphere with 0.8 baroverpressure. The serum bottles (in total 10) were incubated at 37° C.,a rotary speed of 160 rpm and a shaking diameter of 2.5 cm.

To monitor glucose consumption the cultivation of one bottle was stoppedand sampling and HPLC analysis were performed after 0, 3, 4, 5, 7, 8 and8.5 h. After 8.5 h (the glucose concentration was 3.4 g/l) thecultivation was stopped. Aliquots of 0.5 ml cell suspension and 0.5 mlsterile glycerol were filled in cryovials, mixed and stored for 13 h at−20 and afterwards at −80° C. as MCB. The MCB was tested for purity bystreaking a loop of the last cryovial on agar plates for contaminationcontrol and checking in liquid culture (media as described table 8) theproduct spectrum and for contamination (by microscopy).

Consumption of glucose and formation of SA and by-products werequantified via HPLC analyses of the undiluted cell free supernatants ofthe cultivation broth using RI-detection. Broth samples were taken witha sterile syringe through the butyl rubber plug, cell separation wasperformed by filtration (0.22 μm). A 300×7.8 mm I. D. Column AminexHPX-87 H (Biorad) and 5 mm H2SO4 were used as stationary and mobilephase, respectively. The column temperature was 30° C., the flow ratewas 0.5 ml min⁻¹.

One vial of the MCB was used to inoculate a 100 ml-serum bottle with gastight butyl rubber stopper (see above) containing 50 ml of the liquidmedium with 50 g/l glucose. Incubation was performed for 10 h at 37° C.in a shaking incubator (rotary speed: 180 rpm, shaking diameter: 2.5cm). At the end of the cultivation the glucose concentration was 20 g/land the pH around 6.5. Aliquots of 0.5 ml cell suspension and 0.5 mlsterile glycerol were filled in cryovials, mixed and stored at −80° C.as WCB. Purity checks were the same as for the MCB. HPLC conditions werethe same as those described above.

Example 5 Cultivation of Various DD1 Strains on Glycerol or Glycerol andMaltose

The productivity of the mutant strain DD1Δ pfl (LU15348) and DD1Δ pfl Δldh (LU15224) in the presence of gylcerol or glycerol and maltose as acarbon source was further analyzed utilizing the following medium andincubation conditions.

1. Medium Preparation

The composition and preparation of the cultivation medium is asdescribed in the following table 3.

TABLE 3 Medium composition for DD1 cultivation on the substratesglycerol or glycerol and maltose. Compound Concentration [g/l] 1 Bactoyeast extrakt (Becton Dickinson) 10 2 (NH₄)₂SO₄ 2 3 CaCl₂*2H₂O 0.2 4MgCl₂*6H₂O 0.2 5 NaCl 1 6 K₂HPO₄ 3 7 MgCO₃ (Riedel-de Haen 13117) 1 g/gsubstrate 9 NaHCO₃ 8.4 10 substrate varying

Alternative Synthetic Growth Medium

It is favorable to use a synthetic growth medium without complexingredients for the fermentation in order to improve downstreamprocessing and design a synthetic growth medium for cost efficientfermentation.

Medium Preparation

The synthetic growth medium was developed in relation to other syntheticgrowth media for rumen bacteria (Nili and Brooker, 1995, McKinlay et al,2005), previous in house experience with other bacteria and byperforming single omission experiments. Finally, the medium contained 50g/L glucose, 1 g/L (NH₄)₂SO₄, 0.2 g/L CaCl₂*2H₂O, 0.2 g/L MgCl₂*6H₂O, 1g/L NaCl, 3 g/L K₂HPO₄, 1 mg/L nicotinic acid, 1.5 mg/L pantothenicacid, 5 mg/L pyridoxine, 5 mg/L riboflavin, 5 mg/L biotin, 1.5 mg/Lthiamin HCl, 0.26 g/L lysine, 0.15 g/L threonine, 0.05 g/L methionine,0.71 g/L glutamic acid, 0.06 g/L histidine, 0.07 g/L tryptophane, 0.13g/L phenylalanine, 0.06 g/L tyrosine, 0.5 g/L serine, 0.5 g/L glycine,0.5 g/L cysteine, 0.1 g/L β-Alanine, 0.27 g/L alanine, 0.19 g/L valine,0.23 g/L leucine, 0.16 g/L isoleucine, 0.33 g/L aspartic acid, 0.1 g/Lasparagine, 0.13 g/L proline, 0.15 g/L arginine and/or 0.1 g/Lglutamine.

Serum bottles containing 50 mL of synthetic growth medium wereautoclaved with water and 30 g/L MgCO₃ as the buffer system. Glucose,ammonium sulfate and potassium phosphate were sterilized, separately.Ca-, Mg- and Na-chlorides were sterilized together. Vitamins and aminoacids were assembled in various stock solutions and filter sterilized.After cooling down the serum bottles the components were added assterile stock solutions.

2. Cultivations and Analytics

For growing the seed culture one vial of the WCB was used to inoculate a100 ml-serum bottle with gas tight butyl rubber stopper (see above)containing 50 ml of the liquid medium described in table 2 but with 20g/l glucose and a CO₂— atmosphere with 0.8 bar overpressure. Incubationwas performed for a mutant-specific number of hours (table 4) at 37° C.and 160 rpm (shaking diameter: 2.5 cm) The cell suspension wascentrifuged (Biofuge prima R, Heraeus,) with 5000 g for 5 minutes andthe cell pellet was washed and then resuspended in 50 ml medium withouta carbon source and without MgCO₃ to generate a glucose-free inoculum(all steps at room temperature and in the anaerobic chamber).

TABLE 4 Incubation time of various DD1 mutant seed cultures Strain Hoursof incubation LU 13843  8 hrs LU 15050 10 hrs LU 15348 13 hrs LU 1522820 hrs

The main cultures were grown in 100 ml-serum bottles containing 10 mlliquid medium with either 50 g/l glycerol or 50 g/l glycerol and 10 g/lD-maltose and in both cases a CO₂— atmosphere with 0.8 bar overpressure.The quality ‘Glycerol 99%, puriss.’ (Riedel-de Haen, product number:15523-1L-R by Sigma-Aldrich Laborchemikalien GmbH, Seelze, Germany) wasused for all experiments. Inoculation was performed with 1.5 ml of theglucose-free inoculum. The bottles were incubated at 37° C., and 160 rpm(shaking diameter: 2.5 cm).

Consumption of the C-sources and production of carboxylic acids wasquantified via HPLC as described in example 4 after 24 h. As glycerolwas measured the column temperature was adjusted to 50° C. to achieve asufficient separation of SA, lactic acid and glycerol which have similarretention times.

Cell growth was measured by measuring the absorbance at 660 nm (OD₆₀₀)using a spectrophotometer (Ultrospec3000, Amersham Biosciences, UppsalaSweden). Cell concentration defined as gram dry cell weight (DCW) perliter was calculated from the pre-determined standard curve relating theOD₆₀₀ to DCW (1 OD₆₀₀=0.27 g DCW I⁻¹).

3. Results

The results of the cultivation experiments with for different DD1strains are shown in table 5 for the substrate glycerol and table 6 forthe substrate mixture of glycerol and maltose.

TABLE 5 Cultivation of various DD1 strains on glycerol DD1 strainLU13843 LU15348 LU15050 LU15224 tc [h]^(a) 24 24 24 24 ΔC_(Glycerol)[g/l]^(b) −17.3 −25.8 −17.4 −28.6 ΔC_(SA) [g/L]^(c) 19.5 29.9 19.9 36.2ΔC_(LA) [g/L]^(c,h) <0.01 <0.01 <0.01 0.04 ΔC_(FA) [g/l]^(c,h) 0.2 0.050.2 <0.01 ΔC_(AA) [g/l]^(c,h) 1.0 0.6 1.1 0.3 ΔC_(PA) [g/l]^(c,h) 0.3<0.01 0.4 <0.01 ΔC_(MA) [g/l]^(c,h) <0.01 <0.01 <0.01 <0.01 Sum of sideproducts 1.5 0.7 1.7 0.3 SSP [g/l]^(d) SA/SSP [g/g]^(e) 13.0 46.011.7 >100 Ratio SA/FA^(f) 97.5 >100 99.5 >100 Ratio SA/AA^(f) 19.5 49.818.1 >100 STY [g/(l h)]^(g) 0.81 1.24 0.82 1.50 Carbon Yield (YP/S) 1.121.15 1.13 1.26 [g/g]^(g) ^(a)cultivation time. ^(b)consumption ofsubstrate (glycerol, maltose). ^(c)formation of succinic, lactic,formic, acetic, pyruvic and malic acid. ^(d)sum of side products lactic,formic, acetic, pyruvic and malic acid. ^(e)ratio of SA per sum of sideproducts. ^(f)ratio of SA per side product (FA = formic acid; AA =acetic acid). ^(g)space time yield and yield (YP/S) for SA.^(h)Detection limits for acetic acid, lactic acid, malic acid, andformic acid were found to be lower than 0.01 g/l in the given HPLCmethod

In the glycerol-cultivation experiment it is shown that knocking out thepyruvate format lyase gene pfl in a SA producing organism as e.g. DD1leads to significantly higher carbon yield (YP/S) and STY for SA as forthe wildtype when grown on glycerol as a substrate. The carbon yield(YP/S) is increased from 1.12 g/g for the DD1 strain to 1.15 g/g for theΔ pfl mutant strain LU15348.

Other than reported by Lee et al, 2006 or Lin et al 2005 for SAproducing bacteria on glucose, knocking out only the lactatedehydrogenase gene ldhA in DD1 (LU15050) shows no improvement of thetechnical relevant characteristics of this fermentation such as STY ofSA and only minor increase in carbon yield (YP/S). The amount of aceticacid however is even increased if the lactate dehydrogenase enzymeactivity is decreased. Surprisingly and unexpectedly from the analysisof the behavior of strains with the single mutations it was found thatthe mutant strain carrying the combination of the gene mutations in thepfl and the ldh gene the fermentation of glycerol to SA shows evenlarger non additive improvement to the reached carbon yield (YP/S) aswas expected from the single mutations of LU 15050 and LU15348. Thecarbon yield (YP/S) of 1.26 g/g observed is close to the potentialtheoretical carbon yield (YP/S) of 1.28 g/g for the conversion of 1 Molglycerol+1 Mol CO₂ to 1 Mol of SA.

Also the sum of side products (SSP) generated in LU15348 issignificantly decreased while growing on glycerol as no formic acid andless acetic acid is produced. As mentioned above, the SA concentrationis significantly increased compared to wildtype or LU15050. Thisobservation is expressed in the ratios of SA over the arithmetic sum ofside products (SSP) SA:SSP g/g, which exceeds 40 for LU15348 and exceeds100 for LU15224 compared to a level of 10 for LU13843 and LU15050.

In the aforementioned experiment it was found that the STY offermentation of glycerol to SA did not exceed 1.5 g/(l*h) for strainscarrying mutations in the pfl and ldh genes. Therefore, an improvedprocess was developed that showed improved STY values for the productionof SA in an anaerobic fermentation. This process is described in theapplication PCT/EP2008/006714 on the pages 44-46. This process wasadapted for the production of succinate utilizing strains carryingmutations in the genes.

TABLE 6 Cultivation of various DD1 strains on glycerol and maltoseLU13843 LU15348 LU15050 LU15224 tc [h]^(a) 24 24 24 24 ΔC_(Glycerol)[g/L]^(b) −41.2 −48.2 −40 −51.3 ΔC_(Maltose) [g/L]^(b) −11.5 −10.9 −11.3−11.3 ΔC_(SA) [g/L]^(c) 53.2 64.7 51.8 69.8 ΔC_(LA) [g/L]^(c,i) <0.012.5 <0.01 0.1 ΔC_(FA) [g/L]^(c,i) 2.8 <0.01 3.0 <0.01 ΔC_(AA) [g/L]^(c)3.8 0.7 3.6 1.5 ΔC_(PA) [g/L]^(c,i) <0.01 0.6 0.1 0.4 ΔC_(MA)[g/L]^(c,i) 0.05 0.03 0.03 0.1 Σ SP [g/L]^(d) 6.65 3.8 6.73 2.1 SA/SSP[g/g]^(e) 8.0 16.9 7.7 33.2 Ratio SA/FA^(f) 19.11 >100 17.1 >100 RatioSA/AA^(f) 13.9 92.7 14.2 46.2 STY [g/(L h)]^(g) 2.21 2.69 2.15 2.90Yield (YP/S) [g/g]^(g) 1.01 1.09 1.01 1.11 OD₆₀₀ ^(h) 12.9 14.8 16.418.6 DCW [g/L]^(i) 3.5 4 4.4 5 Specific productivity 0.63 0.67 0.49 0.58[g gDCW⁻¹ h⁻¹]^(k) ^(a)cultivation time. ^(b)consumption of substrate(glycerol, maltose). ^(c)formation of succinic, lactic, formic, acetic,pyruvic and malic acid ^(d)sum of side products lactic, formic, acetic,pyruvic and malic acid, ^(e)ratio of SA per sum of side products.^(f)ratio of SA per side product (FA = formic acid; AA = acetic acid).^(g)space time yield and yield (YP/S) for SA. ^(h)Optical density at 600nm, diluting the sample 1:20 with 1M HCl before measuring in aUltrospec2000, Amersham Biosciences, Uppsala Sweden. ^(i)g Biomass asdry cell weight (DCW) ^(h)Specific productivity: g SA per g biomass (drycell weight) per h ^(i)Detection limits for acetic acid, lactic acid,malic acid and formic acid were found to be lower than 0.01 g/l in thegiven HPLC method

Growing DD1 on glycerol simultaneously with another saccharide maltosehas been shown to allow for a higher SA STY and yield (YP/S) and anincreased concentration of side products as compared to using glycerolas the sole substrate (PCT/EP2008/006714 on the pages 44-46).

Comparing LU15348 to the DD1 wildtype shows increased SA amount, STY andcarbon yield (YP/S) if the activity of the Pfl enzyme is decreased.Surprisingly, in contrast to other examples described in the state ofthe art (Lee et al 2006, Lin et al 2005), no growth defect was observedin the mutant strains over the non-mutated strain DD1. This observationis of great technical relevance since good growth of a strain isessential for a technical production process. Cell growth is increasedin all mutants compared to the wildtype. Knocking out either pfl or ldhhas positive effect on growth of the mutated bacterial strain.

Due to lack of detectable formic acid, decreased amount of acetic acidand increased SA concentration, the ratio SA/SSP is increased in mutantstrains containing decreased enzyme activity of Pfl inferred by geneticmutations. However, the side product lactic acid has increased comparedto the wildtype. The double knockout LU15224 has a further increasedyield (YP/S) and STY while LU15050 did not show any improvement incarbon yield (YP/S), STY or the SSP observed.

It is noted that the pfl mutation is necessary and sufficient to improvethe fermentation of glycerol with and without a second saccharidesubstrate over the performance of a wildtype strain in a SA processbased on the metabolization of glycerol. In contrast to this finding aprior art pfl mutation in a wildtype derived strain has not been shownto induce the fermentation of SA (Zhu 2005). Only the combinations ofseveral mutations including pfl and ldh did result in a measurablesuccinic acid production albeit at reduced growth and poor STYperformance (Lin 2005, Lee 2006). The finding of this work teaches theconstruction of an improved process for the fermentative production ofSA consisting of a mutated strain together with a specific process toyield a process with superior performance over the prior art.

Surprisingly, the specific productivity for SA is superior for the pflmutant strain LU15348 over LU15050 and LU15224 carrying mutations in theldh gene and in both ldh and pfl genes. It is known to the expert in thefield that depending on the process a high specific activity of productformation is a desirable characteristic of a technical process.Obviously, the negative effect of knocking out lactate dehydrogenaselowers the specific productivity of LU15348 below the value of thewildtype.

Example 6 Cultivation of LU15348 on Mixtures of Glycerol VariousCarbohydrates

The productivity of the mutant strain LU15348 in the presence ofglycerol and various carbohydrates as a carbon source was analyzedutilizing the following medium and incubation conditions.

1. Medium Preparation, Cultivation and Analytics

The composition and preparation of the cultivation medium is asdescribed in the table 3 of example 5. Cultivation and analytics occuras described in example 5.

The quality ‘Maltodextrin’ (Maldex150, Cat. no.: 50499 by Boom, 7942 JEMeppel, The Netherlands) was used within this experiment. Due to theundefined mixture of saccharide chains with various lengths theconcentration of maltodextrin was not analyzed to full extent byHPLC-analytics. Therefore, maltodextrin content was determinedgravimetrically precisely before being added to the cultivation medium.In order to calculate the lower limit of the achieved theoretical yield(YP/S) it was assumed that all the maltodextrin added to thefermentation had been consumed, being aware that this will allow onlyfor calculation of the lower limit of carbon yield (YP/S) after SAfermentation. More likely, the more exact values of the carbon yield(YP/S) will be higher than the described values due to potentiallyincomplete consumption of the substrate maltodextrin which isundetected.

2. Results

The results of the cultivation experiments for LU15348 are shown intable 7 for the substrate glycerol in cofermentation with variouscarbohydrates as e.g. maltose, maltodextrin or raffinose.

TABLE 7 Cultivation of LU15348 on glycerol in cofermentation withvarious carbohydrates Glycerol + Glycerol + Glycerol + 10 g/L 10 g/L 17g/L LU15348 Maltose Maltodextrin Raffinose Glycerol tc [h]^(a) 24 24 2424 ΔC_(Glycerol) [g/L]^(b) −48.2 −30.5 −37.4 −25.8 ΔC_(Carbohydrate)[g/L]^(b) −10.9 n.a. −1.5 — ΔC_(SA) [g/L]^(c) 64.7 48.4 35.6 29.9ΔC_(LA) [g/L]^(c) 2.5 1.0 0.2 <0.01 ΔC_(FA) [g/L]^(c,i) <0.01 <0.01<0.01 0.05 ΔC_(AA) [g/L]^(c) 0.7 0.4 0.3 0.6 ΔC_(PA) [g/L]^(c,i) 0.6<0.01 0.2 <0.01 ΔC_(MA) [g/L]^(c,i) 0.03 <0.01 <0.01 <0.01 Σ SP[g/L]^(d) 3.8 0.4 0.5 0.7 Ratio SA/SSP [g/g]^(e) 16.9 >100 71.2 46.0Ratio SA/FA^(f) >100 >100 >100 >100 Ratio SA/AA^(f) 92.7 >100 >100 49.8STY [g/(L h)]^(g) 2.69 2.02 1.48 1.24 Yield (YP/S) [g/g]^(g) 1.09 ≧1.02*1.11 1.15 ^(a)cultivation time. ^(b)consumption of substrate (glycerol,maltose). ^(c)formation of succinic, lactic, formic, acetic, pyruvic andmalic acid. ^(d)sum of side products lactic, formic, acetic, pyruvic andmalic acid. ^(e)ratio of SA per sum of side products. ^(f)ratio of SAper side product (FA = formic acid; AA = acetic acid). ^(g)space timeyield and yield (YP/S) for SA. ^(i)Detection limits for acetic acid,lactic acid, malic acid and formic acid were found to be lower than 0.01g/l in the given HPLC method *lower limit of yield (YP/S) due to unknownresidual maltodextrin concentration which was not analyzed.

Cultivating strain LU15348 on glycerol in cofermentation with diversesaccharides such as maltose, a mixture of high molecular saccharide suchas maltodextrin or still another saccharide raffinose, leads to similarresults showing that the simultaneous fermentation of diverse carbonsources including glycerol as one carbon source leads to a number oftechnically relevant improvements of SA production over the state of theart previously described. Examples are increased rates and improvedtotal amounts of glycerol consumed by the process, leading to higher SAtiters as compared to the state of the art. Additionally, the STY isincreased over the control not containing a saccharide. Side productconcentration is generally diminished, except in the case of maltose ascosubstrate, where lactic acid is the increased side product comparedwith the sole glycerol cultivation. The SA yield (YP/S) is similar oronly slightly diminished compared to glycerol as the only substrate.

Summary of the Experiments

-   -   microorganisms that have decreased pfl activity show improved        fermentation on glycerol over the state of art    -   combinations of different carbon sources are efficiently        converted into succinic acid    -   microorganisms that have decreased pfl activity show improved        fermentation on a mixture of glycerol and a saccharide over the        state of art

Conclusion:

The new process for the production of succinic acid (SA) has anexcellent potential for the production of SA and/or SA salts, withcarbon high yield (YP/S) and space time yield as well as very low sideproducts.

In the context of the present invention a bacterial strain DD1 (ID06-614) was deposited with DSMZ (Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany)on Aug. 11, 2006 having the deposit number DSM 18541. In this contextreference is made to the priority European patent application No. EP09152959.4 and EP 09171250.5, wherein the deposit was mentioned thefirst time within the context of the present invention. Furthermore,reference is made to WO 2009/024294, wherein the DD1 strain is describedthe first time including the deposition at DSMZ (Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124Braunschweig, Germany) on Aug. 11, 2006.

The content of the documents cited herein is incorporated by reference.

EXAMPLES Downstream Processing Example 7 Method for Isolation ofSuccinic Acid from Fermentation Broths by Cation Ion Exchange Resin

A fermentation broth neutralized by NH₄OH (25 weight.-%, calculated onthe total weight of the NH₄OH-solution) during the fermentation processwas filtrated. The aqueous, cell free fermentation broth containing 15%(w/w) succinic acid (neutralized as salt) was used for the downstreamprocess.

A cation exchange resin (type Lewatit Monoplus SP 112 from Lanxess; 471ml) was filled in a temperature controlled (50° C.) glass column (bedelevation: 24 cm) as stationary phase and washed with water. After thiswashing step the resin was overflowed in a top down way with the aqueoussuccinic acid solution (156 ml, containing 25 g succinic acid, density:1.069 kg/l). The flow velocity of the solution averaged 33 ml/mincorresponding to a velocity of 4.2 bed volumes (BV) per hour.

Thus a clear and uncolored solution containing approximately 24.9 g freesuccinic acid was obtained in the first fraction (453 ml). The succinicacid concentration of this fraction averaged 5.38 weight.-%.

Beside the index of refraction the pH value and the adsorption (350 nm)of the solution coming out of the column were measured.

The resulting binding capacity of the used strong acid cationicexchanger resin averaged approximately 0.89 equivalents (eq) per literresin.

After the binding process the resin was washed with water (546 ml) andfinally regenerated in the cationic form with 5% hydrochloric acid (919ml; velocity: 66 ml/min) which overflowed the resin bottom up. As a laststep the resin was washed again with water (889 ml).

In addition to the fact, that the resin released the succinic acid, theresin decolorized the broth and a very pure and colorless succinic acidsolution was obtained.

Example 6 Method for Measuring a Break Through Curve with a Strong AcidIon Exchange Resin

Aqueous, cell free fermentation broth containing an amount of 15% (w/w)succinic acid (neutralized as salt) was used for the downstream processafter filtration.

A strong acid ion exchange resin (type Lewatit MonoPlus SP 112 fromLanxess; 689 ml) was filled in a temperature controlled (50° C.) glasscolumn (bed elevation: 97.5 cm) and washed with water. After thiswashing step the resin was overflowed in a top down way with the aqueoussuccinic acid solution (468 ml, containing 75 g succinic acid; density:1.069 kg/l). The flow velocity of the solution averaged 24 ml/mincorresponding to a velocity of 2.1 bed volumes (BV) per hour.

As in example 7, a clear solution (457 ml) containing approximately 53.8g free succinic acid was obtained in the first fraction. The succinicacid concentration of this fraction averaged 11.53 weight-%.

Unlike the assay in example 7, in this case the sampling of the firstfraction was stopped at the moment the cations broke trough. This momentwas detected with the measured pH value which increased suddenly (froman approximately value of 1.4) due to breaking through succinic acidsalt.

The sampled fraction after this clear solution contained succinic acidsalt and has a brown color similar to the original fermentation broth.

In this assay the resulting binding capacity of the used strong acidcationic exchanger resin averaged approximately 1.32 equivalents (eq)per liter resin.

After the binding process the resin was washed with water (678 ml) andregenerated in the cationic form with 5% hydrochloric acid (2034 ml;velocity: 92 ml/min). Finally the resin was washed again with water (824ml).

In addition to the fact, that the resin released the succinic acid, theresin decolorized the broth and a very pure and colorless succinic acidsolution was obtained.

Example 9 Method for Purifying Succinic Acid from Fermentation BrothsFollowed by Concentration and Crystallization of the Resulting DesaltedSolution

Two samples were used for the crystallization step and gained in thesame way as described in example 7.

In each case aqueous, cell free fermentation broth containing an amountof 15% (w/w) succinic acid (neutralized as salt) was used for thedownstream process. The two samples which were purified and desalted byusing a cationic exchange resin (type Lewatit MonoPlus SP 112 fromLanxess) were used for the crystallization step.

The following tables show the volume of fermentation broth, resin andchemicals plus the obtained quantities and capacities in these trials.

Fermenta- Succinic acid Trial tion Dosing concentration number brothresin velocity fraction 1 in fraction 1 34649-069 249 ml 471 ml 33ml/min 462 ml 8.43% 34649-098 249 ml 471 ml 53 ml/min 517 ml 7.57%Succinic Washing HCl (5%) for Washing Resulting Trial acid in waterafter resin water after binding number fraction 1 binding regenerationregeneration capacity 34649- 39.7 g 481 ml 919 ml 928 ml 1.43 eq/l 06934649- 39.9 g 526 ml 919 ml 730 ml 1.44 eq/l 098

Thus two clear and uncolored solutions were obtained as first fractionsand combined to gain one succinic solution (with approximately 76 g freesuccinic acid; difference due to the sampling).

This solution was concentrated by distilling off water to gain 380.2 gsolution with a succinic acid concentration of 20 weight-%. After theconcentration step, the solution was stirred and cooled down. Once thesolution had a temperature of 50° C. it was seeded. Shortly after, thecrystallization began and succinic acid crystals precipitated.

The succinic acid suspension was stirred over night at ambienttemperature and cooled down in an ice/water bath for 1 hour.

The crystals were filtered off in the cold and the cake was washed twotimes with 20 ml ice water each. After the washing step the crystalswere dried in a nitrogen gas flow. Thus 65.2 g colorless succinic acidcrystals with a purity of 99.8% were obtained.

Subsequently, the succinic acid crystals were dried in a fluid be dryer.

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1. A process for the fermentative production of an organic acid or asalt or derivative thereof, which process comprises: a) incubating abacterial strain, capable of utilizing glycerol as a carbon source forthe fermentative production of succinic acid, wherein said strain isgenetically modified so that it comprises a deregulation of itsendogenous pyruvate-formate-lyase enzyme activity, and wherein saidpyruvate-formate-lyase enzyme activity is decreased or switched-off, ina medium comprising an assimilable carbon source and cultivating saidstrain under conditions favouring the formation of the desired organicacid; and b) obtaining said organic acid or salt or derivative thereoffrom the medium.
 2. The process of claim 1, wherein fermentation isperformed at a temperature in the range of about 10 to 60° C. at a pH of5.0 to 9.0 in the presence of carbon dioxide.
 3. The process of claim 1,wherein said organic acid is succinic acid.
 4. The process of claim 1,wherein the assimilable carbon source is selected from glycerol,sucrose, maltose, maltodextrin, D-fructose, D-galactose, D-mannose,lactose, D-glucose, D-xylose, L-arabinose, raffinose decompositionproducts of starch, cellulose, hemicelluloses, and lignocellulose; ormixtures thereof.
 5. The process of claim 4, wherein the carbon sourceis glycerol or a mixture of glycerol and at least one further carbonsource selected from sucrose, maltose, D-fructose, D-galactose, lactose,D-mannose, D-glucose, D-xylose, raffinose, and L-arabinose.
 6. Theprocess of claim 1, wherein the concentration of the assimilable carbonsource is adjusted to a value in a range of 5 to 80 g/L.
 7. A processfor the fermentative production of succinic acid or a salt or derivativethereof, which process comprises: a. incubating a bacterial strain in amedium comprising at least one assimilable carbon source and cultivatingsaid strain under conditions favoring the formation of succinic acid;and b. obtaining said succinic acid or salt or derivative thereof fromthe medium; wherein the process is additionally characterized by atleast one of the following features: i. conversion of at least 25 g/L ofglycerol to at least 25.1 g/L succinic acid, with a yield coefficientYP/S of at least 1.0 g/g; ii. conversion of at least one carbon sourceselected from sucrose, maltose, D-fructose, D-glucose, D-xylose,L-arabinose, D-galactose, D-mannose, raffinose, and/or glycerol tosuccinic acid with a specific productivity yield of at least 0.58 ggDCW-1 h-1 succinic acid; iii. conversion of at least one carbon sourceselected from sucrose, maltose, D-fructose, D-glucose, D-xylose,L-arabinose, D-galactose, lactose, D-mannose, raffinose, and/or glycerolto succinic acid with a space time yield for succinic acid of at least2.2 g/(L h) succinic acid; iv. conversion of at least 25 g/L of at leastone carbon source selected from sucrose, maltose, D-fructose, D-glucose,D-xylose, L-arabinose, D-galactose, lactose, D-mannose, raffinose,and/or glycerol to succinic acid with a space-time-yield for succinicacid of at least 2.2 g/(L h); v. conversion of at least one carbonsource selected from sucrose, maltose, D-fructose, D-glucose, D-xylose,L-arabinose, D-galactose, lactose, D-mannose, raffinose, and/or glycerolto succinic acid with a specific productivity yield of at least 0.6 ggDCW-1 h-1 succinic acid and a space-time-yield for succinic acid of atleast 2.2 g/(L h); vi. production of succinic acid (SA) and sideproducts (SSP) in an SA/SSP proportion of >10:1, or >12.5:1, or >15:1,or >17.5.1, or >20:1, or >25:1, or >30:1, or >33:1, wherein SSPrepresents the sum of side products lactic acid (LA), formic acid (FA),acetic acid (AA), and malic acid (MA), each amount being expressed ing/L; or vii. production of succinic acid (SA) and the side productacetic acid (AA) in an SA/AA proportion of >10:1, or >12.5:1, or >15:1,or >17.5:1, or >20:1, or >25:1, or >30:1 or >50:1, or >75:1, or >90:1,each amount being expressed in g/L.
 8. The process of claim 7, whereinsaid bacterial strain is capable of utilizing glycerol as a carbonsource for the fermentative production of succinic acid, and whereinsaid strain is genetically modified so that it comprises a deregulationof its endogenous pyruvate-formate-lyase enzyme activity, and whereinsaid pyruvate-formate-lyase enzyme activity is decreased orswitched-off, and wherein said strain is derived from a microorganism ofthe family of Pasteurellaceae and comprises the 16S rDNA sequence of SEQID NO: 1 or a sequence having at least 96% sequence homology to SEQ IDNO:
 1. 9. The process of claim 1, performed discontinuously orcontinuously.
 10. A process for the production of succinic acid and/orsuccinic acid ammonium salts comprising a) incubating a bacterialstrain, capable of utilizing glycerol as a carbon source for thefermentative production of succinic acid, wherein said strain isgenetically modified so that it comprises a deregulation of itsendogenous pyruvate-formate-lyase enzyme activity, and wherein saidpyruvate-formate-lyase enzyme activity is decreased or switched-off, ina medium comprising an assimilable carbon source and cultivating saidstrain under conditions favouring the formation of succinic acid and/orsuccinic acid ammonium salts; and b) obtaining said succinic acid and/orsuccinic acid ammonium salts from the medium, wherein fermentation isperformed at a temperature in the range of about 10 to 60° C. at a pH of5.0 to 9.0 in the presence of carbon dioxide, and wherein the pH iscontrolled with ammonia or an aqueous solution thereof, or NH4HCO3,(NH4)2CO3, NaOH, Na2CO3, NaHCO3, KOH, K2CO3, KHCO3, Mg(OH)2, MgCO3,MgH(CO3)2, Ca(OH)2, CaCO3, Ca(HCO3)2, CaO, CH6N2O2, C2H7N, or a mixturethereof.
 11. The process of claim 1, wherein the organic acid and/orsalt thereof are further isolated and/or purified by filtration,crystallization, electrodialysis, and/or cation exchange chromatography.12. The process of claim 1, wherein the organic acid and/or salt thereofis further isolated and/or purified by the following steps: a)filtration, followed by b) cation exchange chromatography, followed byc) crystallization.
 13. The process of claim 11, wherein the cationexchange chromatography is performed with a cation exchange resin in theH+-form carrying sulfonic acid groups having a total capacity of 0.5 to2.0 equivalents/L cation exchange resin.
 14. The process of claim 11,wherein the cation exchange chromatography is performed at a temperatureof from 45 to 60° C.
 15. A process for the production of tetrahydrofuran(THF) and/or 1,4-butanediol (BDO) and/or gamma-butyrolactone (GBL),which comprises a) incubating a bacterial strain, capable of utilizingglycerol as a carbon source for the fermentative production of succinicacid, wherein said strain is genetically modified so that it comprises aderegulation of its endogenous pyruvate-formate-lyase enzyme activity,and wherein said pyruvate-formate-lyase enzyme activity is decreased orswitched-off, in a medium comprising an assimilable carbon source andcultivating said strain under conditions favouring the formation ofsuccinic acid and/or succinic acid ammonium salts; b) obtaining saidsuccinic acid and/or succinic acid ammonium salts from the medium; andeither c1) the direct catalytic hydrogenation of the obtained freesuccinic acid to THF and/or BDO and/or GBL, or c2) the chemicalesterification of the obtained free succinic acid and/or succinic acidsalt to its corresponding di-loweralkyl ester and subsequent catalytichydrogenation of said ester to THF and/or BDO and/or GBL; wherein the pHis controlled with ammonia or an aqueous solution thereof, or NH4HCO3,(NH4)2CO3, NaOH, Na2CO3, NaHCO3, KOH, K2CO3, KHCO3, Mg(OH)2, MgCO3,MgH(CO3)2, Ca(OH)2, CaCO3, Ca(HCO3)2, CaO, CH6N2O2, C2H7N, or a mixturethereof.
 16. A process for the production of pyrrolidones whichcomprises a) incubating a bacterial strain, capable of utilizingglycerol as a carbon source for the fermentative production of succinicacid, wherein said strain is genetically modified so that it comprises aderegulation of its endogenous pyruvate-formate-lyase enzyme activity,and wherein said pyruvate-formate-lyase enzyme activity is decreased orswitched-off, in a medium comprising an assimilable carbon source andcultivating said strain under conditions favouring the formation ofsuccinic acid and/or succinic acid ammonium salts: b) obtaining saidsuccinic acid and/or succinic acid ammonium salts from the medium; andc) chemically converting the succinic acid ammonium salts topyrrolidones, wherein the pH is controlled with ammonia or an aqueoussolution thereof, or NH4HCO3, (NH4)2CO3, NaOH, Na2CO3, NaHCO3, KOH,K2CO3, KHCO3, Mg(OH)2, MgCO3, MgH(CO3)2, Ca(OH)2, CaCO3, Ca(HCO3)2, CaO,CH6N2O2, C2H7N, or a mixture thereof.
 17. The process of claim 4,wherein said glycerol, which is used as the assimilable carbon source,is obtained by ester cleavage of triacylglycerides.
 18. The process ofclaim 17, wherein glycerol is a waste product obtained from themanufacture of bio diesel.
 19. A process for the fermentative productionof an organic fine chemical, which process comprises: a) incubating abacterial strain, capable of utilizing glycerol as the only carbonsource for the fermentative production of succinic acid, wherein saidstrain is genetically modified so that it comprises a deregulation ofits endogenous pyruvate-formate-lyase enzyme activity, and wherein saidpyruvate-formate-lyase enzyme activity is decreased or switched-off, ina medium comprising an assimilable carbon source and cultivating saidstrain under conditions favouring the formation of the organic finechemical; and b) obtaining said organic fine chemical from the medium;wherein the assimilable carbon source comprises glycerol and wherein theorganic fine chemical comprises succinic acid or a salt thereof.
 20. Theprocess of claim 1, wherein said strain is derived from a microorganismof the family of Pasteurellaceae and comprises 1) the 165 rDNA sequenceof SEQ ID NO: 1; 2) a sequence having at least 96% sequence identity toSEQ ID NO: 1; 3) the 235 rDNA sequence of SEQ ID NO: 2; or 4) a sequencehaving at least 95% sequence homology to SEQ ID NO: 2.